CN106119140A - One plant height tolerance Wine brewing yeast strain and construction method thereof - Google Patents
One plant height tolerance Wine brewing yeast strain and construction method thereof Download PDFInfo
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Abstract
The invention discloses plant height tolerance saccharomyces cerevisiae strain and a construction method thereof, described high tolerance yeast strain carries out knocking out obtaining by the promoter partial sequence before the ORF by the encoding gene CYR1 of adenyl cyclase, and knocking out sequence size is 30bp, 60bp, 90bp, 120bp and 150bp respectively.Accurately knocking out of promoter target sequence is realized by fusion DNA vaccine and Yeast Integrating plasmids YIplac211.Build the saccharomyces cerevisiae mutant obtaining CYR1 promoter excalation.Tolerance test proves that the toleration of the yeast mutant of CYR1 promoter sequence disappearance 90bp and 120bp improves, in 40 DEG C of Semen Maydis thick mash fermentation experiments, on the premise of not affecting other fermenting properties, the yeast strain ethanol production of CYR1 promoter deletion 120bp improves 14.6% than parent strain.Remaining without exogenous gene on genome in the yeast that the present invention builds, therefore can be safe be used for produces.
Description
Technical field:
The present invention relates to genetic engineering field, be specifically related to plant height tolerance Wine brewing yeast strain and a construction method thereof.
Background technology:
The raw material that industrial chemicals, organic solvent, disinfectant and the alcoholic drink that ethanol is important is blent.Along with fossil on the earth
The constantly reinforcement ethanol of the increasingly exhausted and global environmental protection consciousness of the energy (oil, coal, natural gas etc.) will become future
One of main regenerative resource.But, the such as stress conditions such as high temperature, hyperosmosis, high concentration of alcohol all can affect yeast
The growth of cell, and then affect the yield of ethanol.During thick mash fermentation, too high being accomplished by of temperature is lowered the temperature with cold water, and this is also
The economic benefit of alcohol production can be reduced.Therefore, improve the high temperature resistance of commercial production saccharomyces cerevisiae, can be from saving cooling
Water aspect improves the economic benefit that bio-ethanol produces.
For regulating the toleration of yeast cells, Ras-cAMP signal path plays pivotal role, CYR1 gene code
Adenyl cyclase is the key enzyme of this path, the synthesis of catalysis cAMP.Research finds, cAMP is as saccharomyces cerevisiae physiological activity
Second message,second messenger, play an important role in terms of regulation cell growth, metabolism and stress resistant.Have been reported that retrotransposition
Sub-Ty element is inserted into the 5 ' of CYR1 gene and holds coding regions or noncoding region can construct the sudden change with multiple stress resistant
Bacterial strain, do so can cause adenyl cyclase reduced activity (about low three times than wild-type strain), and these mutants are the most right
Lethal heat shock has a resistance, and the pressure condition such as ultra-vioket radiation to other, high concentration of alcohol etc. also has resistance.Liu Ying etc.
Research finds that the CYR1 gene knockout of yeast cells can be had higher heat resist ability.
The change of Ras-cAMP signal path activity can affect the toleration of yeast cells.By the expression to CYR1 gene
Regulation and control, it is achieved the yeast cells change to the toleration of stressful environmental.Change promoter intensity and realize gene expression regulation
Important channel.Fusion DNA vaccine can realize the seamless spliced of genetic fragment, and is not selected to be limited by limited restriction enzyme site, from
And the enzyme action repeated during avoiding traditional plasmid construction and attended operation.Utilize fusion DNA vaccine and integrating vector plasmid
Two step integration methods of YIplac211 mediation, on the premise of not remaining marker gene, it is achieved to CYR1 promoter 3 ' terminal sequence
Knock out in various degree, and then build with the Wine brewing yeast strain of CYR1 promoter weakening strength.The high tolerance wine brewing built
Yeast strain can be used safely in commercial production, two step integration methods of fusion DNA vaccine mediation simultaneously can be widely applied to yeast and
He regulates and controls by Microbial promoters, promotes the development that gene is accurately modified.
Summary of the invention:
Present invention solves the technical problem that and be to provide plant height tolerance Wine brewing yeast strain and a construction method thereof.
For solving above-mentioned technical problem, the present invention adopts the following technical scheme that
The high tolerance Wine brewing yeast strain that the present invention provides is the yeast strain to high temperature and ethanol with certain toleration.
The yeast strain that sets out obtaining height tolerance Wine brewing yeast strain is saccharomyces cerevisiae (Saccharomyces cerevisiae)
CICC32315, is stored in Chinese industrial Microbiological Culture Collection administrative center, and the public is available commercially.
Described high tolerance Wine brewing yeast strain in the case of normal growth performance is unaffected, to 55 DEG C of thermal shocks and
8% (v/v) ethanol has certain toleration, and in 40 DEG C of maize raw material high temperature thick mash fermentations ethanol production relative to parent
Bacterial strain improves 14.6%.
Described high tolerance Wine brewing yeast strain specifically can be by starting strain saccharomyces cerevisiae (Saccharomyces
Cerevisiae)-the 150bp of the adenyl cyclase encoding gene CYR1 promoter of CICC32315 to-30bp (ATG=+1)
This sequence traceless knockout realizes.
Said gene traceless knockout can realize using the following method.Involved by each step, concrete operation method is with reference to existing literary composition
Offer report, such as Joseph Sambrook etc., " Molecular Cloning: A Laboratory guide " second edition, Science Press, 1995.
Obtain sudden change ura3 fragment by PCR method, import to saccharomyces cerevisiae by lithium acetate chemical transformation and set out
Bacterial strain, realizes the URA3 gene mutation of starting strain by homologous recombination；Target sequence upstream and downstream is expanded respectively by PCR method
Length is about the homologous sequence fragment of 1.2kb, then by fusion DNA vaccine, upstream and downstream sequence is fused into seamless fragment；Should
Fragment is cloned on Yeast Integrating plasmids YIplac211, it is thus achieved that can carry out integrating the plasmid knocked out；Plasmid is knocked out integrating
In upstream homology arm or downstream homology arm, select suitable single restriction enzyme site, by plasmid enzyme restriction linearisation；Pass through lithium acetate
Chemical transformation, knocks out linearizing integration plasmid and imports in saccharomyces cerevisiae, with the yeast synthetic medium without uracil
Screening occurs the first step to integrate the yeast mutant of restructuring；The generation first step through identifying is integrated the yeast mutation of restructuring
Strain, obtains, through reversely screening containing 5-fluororotic acid synthetic medium flat board, the yeast mutant occurring second step to integrate restructuring；
Obtain starting strain normal URA3 genetic fragment by PCR method, be conducted into there occurs that two steps are integrated the yeast recombinated and dashed forward
In mutant, with the ura3 marker gene of back mutation.
Wine brewing yeast strain of the present invention can be applicable in bio-fuel alcohol production.
Beneficial effect:
1, the present invention provides the Wine brewing yeast strain of a kind of high tolerance, overcomes common saccharomyces cerevisiae raw under stress conditions
Long bad problem.
2, the Wine brewing yeast strain of the high tolerance that the present invention provides is on the premise of keeping excellent fermenting property, adenylic acid
The expression decrease to some degree of the encoding gene CYR1 of cyclase, and then reduce the Ras-cAMP signal of yeast cells
The activity of path so that the toleration of yeast cells changes, the production for biological alcohol fuel established theoretical basis, and
And there is important market value.
3, target sequence knockout technique used in the present invention, it is to avoid yeast tradition knock out during selection markers residual
Problem, meet from clone condition, can safety for commercial production.And this method has in terms of gene accurately modification
Wide application prospect.
Accompanying drawing illustrates:
Fig. 1 is the phenotype checking of AY15a Δ U；
Fig. 2 is the structure schematic flow sheet of recombiant plasmid；
Fig. 3 is the digestion verification figure of recombiant plasmid:
Swimming lane M is DNA maker；1:SalI and BamHI double digestion vector plasmid YIplac211；2-6: be SalI respectively
With BamHI double digestion YIplac211-UD-30, YIplac211-UD-60, YIplac211-UD-90, YIplac211-UD-
120、YIplac211-UD-150；
Fig. 4 is the structure schematic flow sheet of yeast recombinant strain strain；
Fig. 5 is the electrophoresis proof diagram occurring the first step to integrate the bacterial strain recombinated:
Swimming lane M is DNA maker；1:AY15a Δ U is template；2-6: template is respectively AY15a Δ U-U-30, AY15a Δ
U-U-60, AY15a Δ U-U-90, AY15a Δ U-U-120, AY15a Δ U-U-150, CYR1-U-F and YIp-D-F are that checking is drawn
Thing；7:AY15a Δ U is template；8-13: template be respectively AY15a Δ U-U-30, AY15a Δ U-U-60, AY15a Δ U-U-90,
AY15a Δ U-U-120, AY15a Δ U-U-150, YIp-U-R and CYR1-D-R are checking primer；
Fig. 6 is the electrophoresis proof diagram occurring second step to integrate the bacterial strain recombinated:
Swimming lane M is DNA maker；1:AY15a Δ U is template；2-6: template is respectively AY15a Δ U-30, AY15a Δ U-
60, AY15a Δ U-90, AY15a Δ U-120 and AY15a Δ U-150, CYR1P-U and CYR1P-D are checking primer；
Fig. 7 is the sequence verification of yeast recombinant strain strain；
Fig. 8 is parent strain and the growth curve of yeast recombinant strain strain:
Wherein, A: cultivation temperature is 30 DEG C, and B: cultivation temperature is 40 DEG C；
Fig. 9 is the tolerance test result figure of parent strain and yeast recombinant strain strain.
Detailed description of the invention:
The present invention is described below by specific embodiment.Unless stated otherwise, technological means used in the present invention
It is method known in those skilled in the art.It 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 various changes carrying out the material component in these embodiments and consumption or change are also
Belong to protection scope of the present invention.
The structure of embodiment 1:CYR1 promoter excalation Wine brewing yeast strain
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 solid medium contains 2% import agar powder.
According to the Yeast genome data in Genebank and integrated plasmid sequence, devise following primer.
Primer used in table 1 the present embodiment
Note: lowercase alphabet not restriction enzyme site, the overlapping sequence of underscore table not fusion DNA vaccine the primer.
(1) structure of the saccharomyces cerevisiae AY15a Δ U with defective ura3 gene
The Yeast genome using Solarbio company extracts test kit, extracts the genome of laboratory strains W303-1a.
Utilizing primer to URA3-F and URA3-R, with the genome of W303-1a as template, PCR amplification bacterial strain W303-1a suddenlys change ura3 sheet
Section.After test kit reclaims, fragment is imported in parent strain CICC32315 by lithium acetate chemical transformation, logical
Cross the homologous recombination between sudden change ura3 and normal URA3, it is achieved the URA3 gene mutation of reference culture.Bacteria suspension after conversion,
Coat and added on 5-fluororotic acid (5-FOA) and uracil (uracil) yeast synthetic medium (SD) flat board, 30 DEG C of trainings
Support 48h, it is thus achieved that with the saccharomyces cerevisiae AY15a Δ U of defective ura3 gene.The mutant obtained just determines through phenotype checking
Really, AY15a Δ U is the longest on yeast synthetic medium as shown in Figure 1, well-grown on YPD flat board.
(2) integration knocks out the structure of plasmid YIplac211-UD-120
First, with the genome of parent strain CICC32315 as template, use primer pCYR1-U-SalI and rCYR1-U-
Δ 120 obtains the gene order of 1151bp, PCR reaction condition: 95 DEG C of 5min on the left of target sequence pCYR1-120 by PCR；94℃
40s, 53 DEG C of 1min, 72 DEG C of 80s, 30 circulations；72℃10min；Use primer pCYR1-D-Δ 120 and rCYR1-D-simultaneously
The gene order of 1201bp, PCR reaction condition: 95 DEG C of 5min on the right side of BamI amplification target sequence；94 DEG C of 40s, 53 DEG C of 1min, 72 DEG C
80s, 30 circulations, 72 DEG C of 10min, it is respectively designated as U-120 and D-120.Then, with the mixture of U-120 and D-120 as mould
Plate, adds primer pCYR1-U-SalI and rCYR1-U-Δ 120 carries out fusion DNA vaccine, it is thus achieved that seamless fusion fragment.Return through cutting glue
After receiving (test kit), respectively with SalI and BamHI double digestion, finally it is subcloned into the corresponding enzyme action of integrated plasmid YIplac211
In site, integration knocks out plasmid YIplac211-UD-120 and successfully constructs, and deletion sequence size is 120bp, builds flow process such as Fig. 2
Shown in.In order to determine the CYR1 promoter intensity of optimum, construct other four kinds restructuring matter by same plasmid construction method
Grain, respectively YIplac211-UD-30 (deletion sequence size is 30bp), (deletion sequence size is YIplac211-UD-60
60bp), YIplac211-UD-90 (deletion sequence size is 90bp), (deletion sequence size is YIplac211-UD-150
150bp), these five kinds of plasmids are referred to as YIplac211-UD*.Fig. 3 is the digestion verification of recombiant plasmid YIplac211-UD*
Figure.
The employing that above-mentioned fusion DNA vaccine method is known in the art has the primer of spacer end, is formed and has overlapping sequence PCR
Product, is extended by PCR primer overlapping sequence, thus the method that any DNA fragment is coupled together, this technology need not inscribe
The digestion of enzyme and the process of ligase, so that it may realize the Ligation in vitro of DNA fragmentation, this makes integrated plasmid integrate weight through two steps
After group, Yeast genome will not remain external source restriction enzyme site.
(3) structure of CYR1 promoter excalation yeast mutant
BglII enzyme action recombiant plasmid YIplac211-UD*；With lithium acetate chemical transformation by linearizing recombiant plasmid
YIplac211-UD* imports in defective yeast strains A Y15a Δ U, after two steps integrate restructuring, obtains CYR1 gene promoter
The saccharomyces cerevisiae of son 3 ' end portion disappearance, two steps integrate regrouping process such as Fig. 4.
The first step integrates the generation of restructuring, is owing to the analogous parts of the linearization plasmid imported with Yeast genome occurs
Integrate, thus bring whole plasmid into genome.Bacteria suspension after conversion, coats the yeast synthetic medium without uracil
On flat board, 30 DEG C cultivate 48h, it is thus achieved that occur the first step integrate restructuring yeast strain, be respectively designated as AY15a Δ U-U-30,
AY15a Δ U-U-60, AY15a Δ U-U-90, AY15a Δ U-U-120 and AY15a Δ U-U-150, this 5 strain bacterium is referred to as AY15a
ΔU-U*.Use CYR1-U-F and YIp-D-F, YIp-U-R and CYR1-D-R for checking primer, carry out PCR checking screening.When making
When CYR1-U-F and YIp-D-F being carried out PCR checking with primer, because the anneal sequence of downstream primer YIp-D-F exists
On YIplac211 vector plasmid, so yeast strain AY15a Δ U is without specific band, and a step integrates recombinant bacterial strain AY15a
Δ U-U* should have a size to be the band of about 1800bp；When using primer, YIp-U-R and CYR1-D-R is carried out PCR checking
Time because the anneal sequence of forward primer YIp-U-R is on YIplac211 vector plasmid, so yeast strain AY15a Δ U without
Specific band, and a step is integrated recombinant bacterial strain AY15a Δ U-U* and a size should be had to be about 3000bp band.PCR primer
Carry out 0.8% agarose gel electrophoresis result and see Fig. 5.Transformant shows with comparing result, and linearizing plasmid integration arrives
Purpose site.
Integrate restructuring yeast strains AY15a Δ U-U* through screening and the step identified, take a ring and receive 5ml liquid YPD
In culture medium, at 30 DEG C after 200rpm vibration 12h, dilute 10 times of yeast synthesis coated containing 5-fluororotic acid and uracil
On culture medium flat plate, cultivate 48h for 30 DEG C, it is thus achieved that occur second step to integrate the yeast strain of restructuring, after second step integrates restructuring, go out
Existing two kinds of results: one, if integrated between the repetitive sequence that formed of upstream homologous sequence (U), then yeast reverts back to set out
Strains A Y15a；Two, if integrated between the repetitive sequence that formed of downstream homologous sequence (D), then in the middle of all sequences eject,
Realize accurately knocking out of promoter, on target position, do not introduce any exogenous gene simultaneously.Selecting obtained single bacterium colony, employing is drawn
Thing, to CYR1P-U and CYR1P-D, carries out PCR checking, PCR primer stripe size should be respectively 366bp, 336bp, 306bp,
276bp, 246bp and 216bp.Filter out the transformant that promoter target sequence part is accurately knocked out, be respectively designated as AY15a
Δ U-30, AY15a Δ U-60, AY15a Δ U-90, AY15a Δ U-120 and AY15a Δ U-150.PCR primer carries out 2% agar
Sugar Gel electrophoresis results is shown in Fig. 6.Transformant shows with comparing result, and the promoter part of CYR1 obtains striking in various degree
Remove.
The reply of (4) two step recombinant Saccharomyces cerevisiae bacterial strain sudden change ura3 genes
With the method described in (1) in this example, utilize primer to URA3-F and URA3-R, expand AY15a normal URA3 base
Because of fragment, by lithium acetate chemical conversion, to reply the ura3 gene of two step recombinant yeast plant mutants, finally construct the most residual
Stay the traceless knockout bacterial strain of any exogenous gene sequence, be respectively designated as AY15a-30, AY15a-60, AY15a-90, AY15a-
120、AY15a-150。
Extract two steps respectively and integrate the genome of restructuring yeast strains, use primer that CYR1P-F and CYR1P-R is carried out
PCR reacts.PCR product checks order through Hua Da genome company, and sequencing result announces sequence alignment with NCBI, the result such as figure
7.The promoter of the CYR1 of AY15a-30, AY15a-60, AY15a-90, AY15a-120, AY15a-150 as can be seen from Figure 7
Deletion condition is respectively 30bp, 60bp, 90bp, 120bp, 150bp.
Embodiment 2:CYR1 promoter excalation Wine brewing yeast strain growth performance measures and tolerance test
(1) growth performance of CYR1 promoter excalation Wine brewing yeast strain measures
Measure parent strain CICC32315 and transformant AY15a-30 thereof, AY15a-60, AY15a-90, AY15a-120,
AY15a-150 growth performance under the conditions of 30 DEG C and 40 DEG C, result such as Fig. 8.Result shows, strikes the part of CYR1 promoter
Except having not significant impact yeast on the premise of the growth performance of 30 DEG C, the growth performance of the yeast cells at 40 DEG C all obtains
A certain degree of raising.
(2) tolerance test of CYR1 promoter excalation Wine brewing yeast strain
Utilize dilution point plate measuring parent strain CICC32315 and transformant AY15a-30 thereof, AY15a-60,
The toleration change of AY15a-90, AY15a-120, AY15a-150.
Condition of culture:
One-level: take a ring bacterium mud and be connected in 5mLYPD liquid, 30 DEG C, 180rpm, cultivates 12h.
Two grades: take 200 μ L one-level bacterium solution and be connected in 5mLYPD liquid, 30 DEG C, 180rpm, cultivates 6-8h to yeast cells
Logarithmic (log) phase.
The OD of the yeast cells of regulation logarithmic (log) phase600=1.2, it is used for doing tolerance test.To monoploid parent and conversion thereof
Son respectively takes 200 μ L and tests for thermal shock, 55 DEG C of thermal shock 3min, and is diluted to 10-4, then respectively take 3 μ L not from different dilution factors
Do the bacterium solution of thermal shock experiment and do thermal shock experiment bacterium solution and put respectively and be connected on YPD flat board；Respectively take 3 μ L from different dilution factors again not do
The bacterium solution of thermal shock experiment is put on the YPD flat board being connected to containing 260g/L glucose or 8% (v/v) ethanol respectively, cultivates 3 for 30 DEG C
My god, take pictures.Result such as Fig. 9.Result display AY15a-90 and AY15a-120 not only shows good heat resistance, and right
The toleration of 8% (v/v) ethanol is also improved to some extent.
Case study on implementation 3: strains A Y15a-90 and the experiment of AY15a-120 maize raw material thick mash fermentation
(1) AY15a-90 and AY15a-120 tests with the maize raw material thick mash fermentation of parent strain
Parent strain CICC32315 and transformant AY15a-90 and AY15a-120 are carried out Semen Maydis thick mash the most simultaneously and send out
Ferment is tested, fermentation technology route map: Semen Maydis powder → immersion → liquefaction → saccharifying → cool down → connect bacterium → fermentation → steaming wine → mensuration
Index；
Process conditions:
Soaking conditions: 60～70 DEG C, impregnates 20min；Liquefaction condition: 85～90 DEG C, adds Thermostable α-Amylase, liquefaction
90min；Saccharifying condition: 55～60 DEG C, adds saccharifying enzyme, saccharifying 20min；
Dispensing: Semen Maydis powder 60g, water 180mL, Thermostable α-Amylase 2 × 105U/mL, 30 μ L, saccharifying enzyme 200U/mL, 90
μ L, 2.5 × 103U/mL acid protease 1.2mL；Nutritive salt 1mL (MgSO4 150g/L、KH2PO475g/L, carbamide 81g/L,
Filter, 4 DEG C of preservations)；Inoculum concentration: 7.5%, places 12h, places into 40 DEG C, ferment 3 days, measure each bacterium after fermentation ends for 30 DEG C
Fermenting property (the CO of strain2Weightlessness, ethanol production, residual sugar)；The results are shown in Table 2.
Table 2 strain fermentation results contrast
Note: shown data are the result of three parallel tests.
Table 2 shows, under the conditions of the high temperature thick mash fermentation at 40 DEG C, and transformant AY15a-90 and the CO of AY15a-1202Lose
Weight and ethanol production the most relatively parent strain increase, and the ethanol production of AY15a-90 with AY15a-120 divides relative to parent strain
Do not improve 14.0% and 14.6%, illustrate that AY15a-120 is an advantage over the high tolerance Wine brewing yeast strain of AY15a-90.
Claims (6)
1. the high tolerance Wine brewing yeast strain selected, is to weaken adenyl cyclase coding base in saccharomyces cerevisiae starting strain
Because the promoter intensity of CYR1 obtains.
2. high tolerance Wine brewing yeast strain as claimed in claim 1, it is characterised in that described starting strain is saccharomyces cerevisiae
(Saccharomyces cerevisiae)CICC32315。
3. high tolerance Wine brewing yeast strain as claimed in claim 1, it is characterised in that described saccharomyces cerevisiae has normal raw
55 DEG C of thermal shocks and 8% (v/v) ethanol are had certain toleration by long performance, and at 40 DEG C of maize raw material high temperature thick mash
In fermentation, ethanol production improves 14.6% relative to parent strain.
4. the construction method of high tolerance Wine brewing yeast strain as claimed in claim 1, it is characterised in that described construction method is
The two step gene integration methods mediated by fusion DNA vaccine technology and integrating vector plasmid YIplac211, are cyclized encoding adenovirus thuja acid
Enzyme gene C YR1 promoter 3 ' end-150bp realizes to this sequence traceless knockout of-30bp (ATG=+1).
5. the construction method of high tolerance Wine brewing yeast strain as claimed in claim 4, it is characterised in that concrete steps include:
Obtain sudden change ura3 fragment by PCR method, import to saccharomyces cerevisiae starting strain by lithium acetate chemical transformation, pass through
Homologous recombination realizes the URA3 gene mutation of starting strain；Expand target sequence upstream and downstream length respectively to be about by PCR method
The homologous sequence fragment of 1.2kb, then by fusion DNA vaccine, is fused into seamless fragment by upstream and downstream sequence；This fragment is cloned
To Yeast Integrating plasmids YIplac211, it is thus achieved that can carry out integrating the plasmid knocked out；Integrating the upstream homology knocking out plasmid
In arm or downstream homology arm, select suitable single restriction enzyme site, by plasmid enzyme restriction linearisation；By lithium acetate chemical conversion
Method, knocks out linearizing integration plasmid and imports in saccharomyces cerevisiae, screen with the yeast synthetic medium without uracil and occur
The first step integrates the yeast mutant of restructuring；The generation first step through identifying is integrated the yeast mutant of restructuring, through containing
5-fluororotic acid synthetic medium flat board reversely screens and obtains the yeast mutant occurring second step to integrate restructuring；Use PCR method
Obtain starting strain normal URA3 genetic fragment, be conducted into there occurs in the yeast mutant that two steps integrate restructuring, with
The ura3 marker gene of back mutation.
6. high tolerance Wine brewing yeast strain application in alcohol production as claimed in claim 2.
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| CN107603896A (en) * | 2017-08-22 | 2018-01-19 | 上海理工大学 | The Wine brewing yeast strain of one plant height ethanol-tolerant high fermentation rate |
| 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 |
| CN108865912A (en) * | 2018-06-22 | 2018-11-23 | 华南理工大学 | A kind of methanol height tolerance pichia pastoris engineered strain and its construction method |
| CN109576251A (en) * | 2018-11-07 | 2019-04-05 | 河南农业大学 | Corn adenyl cyclase ZmRPP13-LK3, its encoding gene and its application |
| US20210198679A1 (en) * | 2019-12-27 | 2021-07-01 | Tianjin University Of Science And Technology | Saccharomyces cerevisiae strain with high yield of ethyl butyrate and construction method and application of saccharomyces cerevisiae strain |
| CN117264791A (en) * | 2023-03-19 | 2023-12-22 | 复旦大学 | Kluyveromyces marxianus engineering strain for efficiently expressing exogenous proteins and application thereof |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN107603896A (en) * | 2017-08-22 | 2018-01-19 | 上海理工大学 | The Wine brewing yeast strain of one plant height ethanol-tolerant high fermentation rate |
| CN108865912A (en) * | 2018-06-22 | 2018-11-23 | 华南理工大学 | A kind of methanol height tolerance pichia pastoris engineered strain and its construction method |
| CN109576251A (en) * | 2018-11-07 | 2019-04-05 | 河南农业大学 | Corn adenyl cyclase ZmRPP13-LK3, its encoding gene and its application |
| CN109576251B (en) * | 2018-11-07 | 2019-09-27 | 河南农业大学 | Corn adenyl cyclase ZmRPP13-LK3, its encoding gene and its application |
| US20210198679A1 (en) * | 2019-12-27 | 2021-07-01 | Tianjin University Of Science And Technology | Saccharomyces cerevisiae strain with high yield of ethyl butyrate and construction method and application of saccharomyces cerevisiae strain |
| US11746353B2 (en) * | 2019-12-27 | 2023-09-05 | Tianjin University Of Science And Technology | Saccharomyces cerevisiae strain with high yield of ethyl butyrate and construction method and application of Saccharomyces cerevisiae strain |
| CN117264791A (en) * | 2023-03-19 | 2023-12-22 | 复旦大学 | Kluyveromyces marxianus engineering strain for efficiently expressing exogenous proteins and application thereof |
| CN117264791B (en) * | 2023-03-19 | 2024-06-18 | 复旦大学 | Kluyveromyces marxianus engineering strain for efficiently expressing exogenous proteins and application thereof |
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