CN109136254A - A kind of seamless gene knockout method of efficient saccharomyces cerevisiae and its application - Google Patents

Abstract

The method for efficient seamless knockout that the invention discloses a kind of in saccharomyces cerevisiae, and by being optimized to positive homologous sequence, galactose concentration and the galactolipin induction time in the seamless gene knockout system, the probability of second step homologous recombination reaches 6.86 × 10^‑4.It is starting strain with the monoploid α 5 of saccharomyces cerevisiae AY15, BAT2 gene is target gene, it realizes to the efficient seamless knockout of Wild Saccharomyces cerevisiae strain gene BAT2, it is tested by liquor fermentation, it is transformed that the normal propyl alcohol of bacterial strain, isobutanol is compared with isoamyl alcohol content parent strain reduces 20.32%, 47.85% and 23.14% respectively, has achieved the purpose that yield of higher alcohol.This method can be widely applied to yeast and the genetic modification of other microorganisms, since mutant strain obtained does not remain any foreign gene, can safety be used for industrial production, for directly in industrial strain progress gene knockout provide beneficial reference.

Description

A kind of seamless gene knockout method of efficient saccharomyces cerevisiae and its application

Technical field:

The present invention relates to genetic engineering fields, and a kind of efficient seamless gene knockout side is specifically constructed in saccharomyces cerevisiae Method simultaneously constructs the Wine brewing yeast strain of one plant of yield of higher alcohol with this method.

Background technique:

Saccharomyces cerevisiae is a kind of typical eukaryocyte model and important model organism, its characteristic includes: that growth is numerous Grow that fast, the metabolism period is short, is easily isolated and cultivate, these characteristics make saccharomyces cerevisiae more convenient for progress genetic engineering and heredity Learn research, " Escherichia coli " referred to as in eucaryote.With molecular biology and technique for gene engineering continuous development and It constantly updates, the breeding method of saccharomyces cerevisiae is modern directed gene Engineering Breeding via initial natural breeding development. Gene knockout (gene knockout) is a kind of genetic engineering technology to grow up late 1980s, is by appropriate Method make the specific gene delection of body or inactivation, with achieve the purpose that pointed decoration transformation chromosome on a certain gene one Kind technology.But the concern food-safe with the public, the bacterial strain constructed by traditional genetics method, due to residual non- The foreign gene of body, production application are restricted.Therefore become mesh using the specific gene that seamless knockout technology knocks out body The developing direction of preceding genetic breeding.

The seamless modification of saccharomycete is initially in order to remove selection markers, to carry out polygenes behaviour in single bacterial strain Make.Removing selection markers, there are mainly two types of methods, and one is the knockout systems using recombinase-mediated, such as Cre/Loxp system System, Cre recombinase, which can mediate, occurs specificity recombination between two sites LoxP, the gene elmination between two sites.Pass through Segment with two sites LoxP in the same direction and selection markers is integrated on genome by first time chemical conversion；Then pass through Second of chemical conversion imports the plasmid with coding Cre recombination enzyme gene, cuts off two sites using the expression of Cre recombinase Between all sequences, to complete the removal of selection markers.2004, which was applied in yeast by Iwaki et al., realized The polygenes of yeast is knocked out.Cre/loxP method has very high efficiency, but can remain an external source on chromosome Sequence (site LoxP), when carrying out polygenes knockout, the remaining site LoxP increases the possibility of chromosomal rearrangement.It is another Kind method is then to utilize the homologous recombination knocked out in element between direct repetitive sequence (hisG), it is necessary first to which building has The plasmid of " hisG-URA3-hisG ", then directly obtains knockout element using long primer PCR, knocks out purpose base by conversion Because after, then reversed screening, selection markers are removed using the homologous recombination between direct repetitive sequence (hisG).2012, Dong Et al. application the method achieve the seamless knockout of yeast genes, but this method is in the probability of induction second step homologous recombination Random, the transformant more than one of generation, so that the probability for finally obtaining ideal transformant is lower.At the beginning of 2013, Yi Zhongquan New artificial endonucleases clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas)) occur, it is mainly based upon a kind of acquired immune system transformation of bacterium It forms, its main feature is that production is simple, at low cost, effect is efficient.2014, Bao etc. was for the first time using CRISPR-Cas system to wine Brewer yeast realizes that one step of polygenes knocks out.But the guide RNA (sg RNA) designed in the system is instructing Cas albumen and gene Group can introduce unexpected gene mutation, i.e. undershooting-effect, seriously constrain when being identified with non-target spot DNA sequence dna mispairing The extensive use of the technology.In short, there is still a need for a kind of efficient seamless gene knockout methods for this field.

Higher alcohol is the alcohol such as important metabolite and white wine, grape wine in saccharomyces cerevisiae alcoholic fermentation process drink Main flavor substance in material, content number and various alcohol between ratio have important influence to the flavor of wine, fit The higher alcohol of amount and the component ratio of coordination can assign wine special fragrance, while set off the fragrance of ester output, assist the mouthfeel of wine It adjusts, is soft.And higher alcohol too high levels can make wine generate different miscellaneous taste, influence the flavor and quality of wine, and higher alcohol is in human body Interior oxidation rate is slower than ethyl alcohol, is significantly larger than ethyl alcohol to the toxic action of human body.Therefore, it is modern for controlling advanced alcohol content An important index in brewing process.In saccharomyces cerevisiae alcoholic fermentation process, there are two higher alcohol metabolic pathways, respectively Glycometabolism route of synthesis (Harris approach) and amino acid catabolic pathway (Ehrlich approach).In amino acid catabolic In approach, the first step of saccharomyces cerevisiae branched-chain amino acid catabolism is transamination.Show that BAT2 gene is compiled according to research reports The branched-chain amino acid transaminase of code plays very important work in the higher alcohol especially generating process of isobutanol and isoamyl alcohol With.Therefore, in order to reduce the production quantities of fermentation process Higher Alcohols, the amino acid transaminase having a major impact to higher alcohol is knocked out Encoding gene BAT2 will be an effective approach.

Therefore, the present invention will construct in saccharomyces cerevisiae a kind of high using amino acid transaminase encoding gene BAT2 as target gene The seamless knockout method of effect, this method can be widely applied to yeast and the genetic modification of other microorganisms, for directly in industrial bacterium Gene knockout is carried out in strain provides beneficial reference.The yield of higher alcohol mutant strain of building can be used safely in industrial production, Meet requirement of the saccharomyces cerevisiae application related fields to yeast.

Summary of the invention:

The technical problem to be solved by the present invention is to provide a kind of methods of efficient seamless gene knockout of saccharomyces cerevisiae, and with should Method constructs the Wine brewing yeast strain of one plant of yield of higher alcohol.

In order to solve the above technical problems, the present invention adopts the following technical scheme:

A kind of seamless gene knockout method of efficient saccharomyces cerevisiae is bacterium germination with the monoploid α 5 of saccharomyces cerevisiae AY15 Strain, BAT2 gene are target gene, realize the seamless knockout to gene BAT2.

Yield of higher alcohol yeast strain constructed by the present invention is to starting strain saccharomyces cerevisiae (Saccharomyces Cerevisiae) it is next real to carry out seamless knockout by the whole amino acid transaminase encoding gene BAT2 in CICC32315 monoploid α 5 It is existing.Specially saccharomyces cerevisiae H α 5 (500) Δ H.

Its Gene ID of the BAT2 gene are as follows: 853613, it is as shown in the table for nucleotide sequence.

Said gene seamless knockout can be realized using the following method.Concrete operation method involved by each step refers to existing text Report, such as JosephSambrook are offered, " Molecular Cloning:A Laboratory guide " second edition, Science Press, 1995.

HERP1.0 segment is amplified with PCR method, which is cloned into YEp352 plasmid, constructs YHERP1.0 plasmid； Being expanded respectively with PCR method and knocking out target gene BAT2 upstream and downstream length is 500bp and the homologous sequence segment of 683bp, so Seamless fusion segment is obtained by fusion DNA vaccine afterwards；The segment is cloned on plasmid YHERP1.0, recombinant plasmid is obtained YHERP1.0(500)；Expand the segment knocked out on target gene upstream 597bp and YHERP1.0 (500) respectively with PCR method；It is logical Peracetic acid lithium conversion method imports the two segments in saccharomyces cerevisiae simultaneously, carries out the first step by YPGly+AF screening and culturing medium The screening of integration obtains the mutant strain containing resistant gene；With 30 DEG C of galactolipin induced medium, 180rpm cultivates dilution for 24 hours It is coated on after 100 times on the synthetic media plate containing FdUrd, 30 DEG C of culture 36h, it is whole to obtain second step Close the Wine brewing yeast strain of recombination.

The saccharomyces cerevisiae knocks out sieve under growth performance and the unaffected situation of fermenting property, in second of integration recombination The probability for selecting labeling process is 6.86 × 10^-4。

Wine brewing yeast strain (H α 5 (500) Δ H) of the present invention can be used in liquor production.

Invention also provides the optimization for induced medium, the content pair of galactolipin in induced medium has been probed into Its second step integrates the influence such as table 1 of recombination probability, and operating process is as follows:

1, the content of galactolipin is equipped with 0.1g/100mL, 0.5g/100mL, 1g/100mL, 2g/ altogether in induced medium Eight gradients of 100mL, 3g/100mL, 4g/100mL, 5g/100mL, peptone 2g/100mL, yeast extract 1g/100mL.

2, H α 5 (500) mutant strain is subjected to Fiber differentiation for 24 hours with gala sugar culture-medium.Coating contains 5- after 100 times of dilution The synthetic media plate of fluoro- 2 '-BrdU.

3, the probability of 1 second step of table integration recombination

The positive effect of the present invention is as follows:

1, the present invention provides a kind of method of seamless gene knockout of efficient saccharomyces cerevisiae, overcomes in traditional gene knockout Selection markers residual, the inconvenient difficulty for carrying out polygenes knockout.The invention simultaneously can be not only used for the function of research yeast genes Can and metabolic mechanism, and since mutant strain obtained does not remain any foreign gene, can safety be used for industrial production.

2, low-yield higher-alcohol saccharomyces cerevisiae provided by the invention knocks out ammonia under the premise of keeping good fermenting property completely Base acid transaminase encoding gene BAT2 is tested by corn thick mash liquor fermentation, the normal propyl alcohol, isobutanol and isoamyl of bacterial strain is transformed Alcohol content is significantly reduced, and has achieved the purpose that yield of higher alcohol, to brew excellent in flavor and being more advantageous to healthy wine Smart beverage has established theoretical basis.

Detailed description of the invention:

Fig. 1 is the building flow diagram of recombinant plasmid YHERP1.0 (500).

Fig. 2 is building and the verifying electrophoretogram of recombinant plasmid YHERP1.0 and YHERP1.0 (500).

Fig. 3 is the two steps integration recombination flow diagram during seamless knockout.

Fig. 4 is the electrophoresis proof diagram that the bacterial strain H α 5 (500) of first step integration recombination occurs.

Fig. 5 is 5 (500) Δ H electrophoresis proof diagram of bacterial strain H α that second step integration recombination occurs.

Fig. 6 is the part sequencing comparison chart for integrating 5 (500) Δ H target gene position recombinant bacterial strain H α, and wherein Fig. 6 a is to knock out Context comparison, Fig. 6 b are the sequencing result of target position 40bp after knocking out.

Fig. 7 is the growth curve of H α 5 (500) Δ H and α 5.

Specific embodiment:

The present invention is described below by specific embodiment.Unless stated otherwise, technological means used in this experiment It is method well known to the technical staff of field.In addition, embodiment is interpreted as illustrative, to be not intended to limit the present invention model It encloses, 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 hair Under the premise of bright spirit and scope, the various changes or change of material component and dosage progress in these embodiments are also belonged to In protection scope of the present invention.

Embodiment 1

The building of seamless knockout amino acid transaminase encoding gene BAT2 saccharomyces cerevisiae

Starting strain used in this example is the monoploid α 5 of CICC32315.(the segment HERP1.0 is shown in source William G.Alexander, Drew T.Doering and Chris Todd Hittinger, High-Efficiency Genome Editing and Allele Replacement in Prototrophic and Wild Strains of Saccharomyces.Genetics,Vol.198,859–866 November 2014).The bacillus coli DH 5 alpha is purchased from Takara company.The YPD culture medium is general complete medium；The screening and culturing medium YPGly+AF ingredient is 5% Glycerol, 2% peptone, 1% yeast extract, 200mg/mL amethopterin, 5mg/mL sulfanilamide (SN), 5 μ g/mL thymidines, 50 μ g/ ML hypoxanthine；Yeast synthetic media (SC) ingredient be 2% glucose, 0.17%YNB, 0.5% ammonium sulfate, completely Amino acid mixture solution, solid medium contain 2% import agar powder.

According to the Yeast genome data and YEp352 plasmid sequence in Genebank, devises and respectively draw in following embodiments Object.

Used primer in 2 the present embodiment of table

Note: underscore adds italic to indicate restriction enzyme site, and italic indicates the overlap of fusion DNA vaccine the primer, underscore Indicate the restriction enzyme site of addition.

PCR amplification system used in 3 the present embodiment of table

Fusion DNA vaccine system used in 4 the present embodiment of table

(1) building of plasmid YHERP1.0

The building process of plasmid is as shown in Figure 1；

Firstly, extracting the genome of yWH245 using the Yeast genome extracts kit of Solarbio company.Using drawing Object YH1.0 (EcoRI)-F and YH1.0 (SphI)-R, using the genome of yWH245 as template, in PCR amplification bacterial strain yWH245 HERP1.0 segment, is recycled by kit；Double digestion, gel extraction are carried out to plasmid YEp352 with EcoRI and SphI restriction endonuclease Digested plasmid；With the Clone of VazymeII One Step Cloning Kit is by digested plasmid and HERP1.0 segment It is attached and is transferred to DH5 α competence, plasmid YHERP1.0 is constructed successfully.

(2) building of recombinant plasmid YHERP1.0 (500)

The building of recombinant plasmid YHERP1.0 (500) is as shown in Figure 1

The Yeast genome extracts kit for using Solarbio company first, extracts the base of the monoploid α 5 of CICC32315 Because of group.BAT2 upstream region of gene 500bp is expanded using primer BAT2-A (KpnI)-F and BAT2-A-R；Primer BAT2-B-F is used simultaneously BAT2 downstream of gene 683bp is expanded with BAT2-B (KpnI)-R, names BAT2-U and BAT2-D respectively.Then with BAT2-U and The mixture of BAT2-D is template, and primer BAT2-A (KpnI)-F and BAT2-B (KpnI)-R is added and carries out fusion DNA vaccine, obtains nothing Seam fusion segment, is named as BAT2-UD.After gel extraction (kit), digestion is carried out with KpnI, is subcloned into plasmid In the corresponding restriction enzyme site of YHERP1.0, and connect with primer AHB-B-U and BAT2-B (KpnI)-R verifying single endonuclease digestion it is positive and negative, Recombinant plasmid YHERP1.0 (500) is constructed successfully.

Fig. 2 is building and the verifying electrophoretogram of recombinant plasmid YHHERP1.0 and YHHERP1.0 (500): swimming lane 1 is HERP1.0 (3159bp) electrophoresis result；Swimming lane 2 is EcoRI and SphI digestion YEp352 plasmid (5100bp) electrophoresis result；Swimming lane 3 be YHERP1.0 plasmid electrophoresis result: swimming lane 4 is YEp352 plasmid electrophoresis result；Swimming lane 5 is KpnI digestion YHERP1.0 plasmid (8300bp) electrophoresis result；Swimming lane 6 is BAT2-U₁D segment (1183bp) electrophoresis result；Swimming lane 7 is YHERP1.0 (500) plasmid Electrophoresis result；Swimming lane 8 is positive and negative (1260bp) electrophoresis result of YHERP1.0 (500) single endonuclease digestion connectivity verification；Swimming lane 9 is EcoRI Digestion YHERP1.0 (500) plasmid (3800bp and 5700bp) electrophoresis result；Swimming lane M is 15000 DNA Ladder maker.

Above-mentioned fusion DNA vaccine method is well known in the art using the primer with spacer end, and being formed has overlap PCR Product is extended through PCR product overlap, so that any DNA segment is connected method, this technology does not need restriction endonuclease Digestion and ligase processing, so that it may realize the Ligation in vitro of DNA.

(3) in saccharomyces cerevisiae amino acid transaminase encoding gene (BAT2) knockout

With 597bp on the left of primer BAT2-A-F and BAT2-A-RF amplification BAT2,635bp long fragment is obtained, is named BAT2-A—SF；It is expanded on YHERP1.0 (500) with primer BAT2-B-FR and BAT2-B (KpnI)-R simultaneously and obtains 4700bp The segment of left and right length is named as RS-HERP1.0-BAT2-UD；With lithium acetate transformation method by segment BAT2-A-SF with RS-HERP1.0-BAT2-UD is imported in Wine brewing yeast strain α 5, after the integration recombination of two steps, obtains seamless knockout BAT2 Saccharomyces cerevisiae, two step regrouping process such as Fig. 3.

The generation of first step integration recombination is since two segments of importing and the analogous parts generation of Yeast genome are whole It closes, and can also be integrated between two segments there are analogous parts, so that whole fragment be made to be integrated into the genome of α 5.Conversion Suspension afterwards is coated on YPGly+AF screening and culturing medium plate, 30 DEG C of culture 7d, is obtained and is occurred what the first step entirely recombinated Yeast strain H α 5 (500).Resulting single colonie is selected at random, uses AHB-A-U and AHB-A-D for the verifying of integration site upstream Primer, AHB-B-U and AHB-B-D are that primer is verified in integration site downstream, carry out bacterium colony PCR screening.Fig. 4 is that the generation first step is whole Close the electrophoresis proof diagram of the bacterial strain H α 5 (500) of recombination: swimming lane M is DL5000 DNA Ladder maker；Swimming lane 1,2 makes respectively Primer PCR verification result is verified with upstream, 1 template of swimming lane is H α 5 (500), and 2 template of swimming lane is α 5；Swimming lane 3,4 is respectively under use Trip verifying primer PCR verification result, 3 template of swimming lane are H α 5 (500), and 4 template of swimming lane is α 5.

By screening and identifying the recombinant Saccharomyces cerevisiae bacterial strain H α 5 (500) of acquisition, a ring is taken to be connected to the induction of 5mL galactolipin In culture medium, 30 DEG C, 180rpm is cultivated for 24 hours, and it is flat that 100 times of dilution is coated on the synthetic media containing FdUrd On plate, 30 DEG C of culture 36h obtain the Wine brewing yeast strain of second step integration recombination.Obtained single colonie is selected at random, is used AHB-A-U and AHB-B-D primer carries out bacterium colony PCR screening, and the probability of verified second integration recombination can achieve 6.86 × 10^-4, can fast and efficiently accomplish the recycling of selection markers, convenient for utilizing again, and achieve the purpose that seamless knockout.Fig. 5 is The electrophoresis proof diagram of 5 (500) Δ H of bacterial strain H α of second step integration recombination occurs: swimming lane M is DL5000 Ladder maker；Swimming Road 1 is the verification result that PCR is carried out using primer AHB-A-U and AHB-B-D, and 1 template of swimming lane is H α 5 (500) Δ H.

In order to further verify the sequence situation for knocking out target position, the gene of 5 (500) Δ H of seamless knockout bacterial strain H α is extracted Group carries out PCR amplification using primer AHB-A-U and AHB-B-D, and the segment for obtaining 2605bp is sent to be sequenced in Huada gene company, Sequencing result such as Fig. 6.Fig. 6 a is to knock out context comparison, and Fig. 6 b is the sequencing result of target position 40bp after knocking out, other knots Fruit does not provide, can determine that seamless knockout gene BAT2 Wine brewing yeast strain constructs successfully.

Embodiment 2

The measurement of seamless knockout BAT2 Wine brewing yeast strain H α 5 (500) Δ H and 5 growth curve of α

The single colonie of 5 (500) Δ H of picking H α and α 5 is inoculated in 50mLYEPD fluid nutrient medium, and 30 DEG C, 180rpm oscillation Culture for 24 hours, takes the bacterium solution for expanding and cultivating to be inoculated in 3 bottles of 50mL YEPD culture mediums according to the inoculum concentration of 1:100, similarity condition Lower shaken cultivation, and after culture 0h, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h takes thallus culture suspension 0.5mL, 10000rpm to be centrifuged 1min, and bacterium mud then is resuspended with deionized water.It is corrected with deionized water The zero point of 7200 type visible spectrophotometers, the colorimetric estimation bacterium solution OD at wavelength 600nm₆₀₀Value.With the suction of each time point bacterium solution Light shading value (OD₆₀₀) it is ordinate, using incubation time as abscissa, the growth curve of each bacterial strain is drawn out, growth curve is as schemed 7, from growth curve it can be seen that H α 5 (500) Δ H is with 5 growth performance of starting strain α, there is no difference.

Embodiment 3

The corn thick mash fermentation of 5 (500) Δ H of seamless knockout BAT2 Wine brewing yeast strain H α and α 5 is tested

Recombinant bacterial strain H α 5 (500) Δ H and starting strain α 5 is subjected to corn thick mash fermentation experiment simultaneously respectively, work of fermenting Skill route map:

Corn flour immersion → liquefaction → saccharification → plus bacterium fermentation → weighing weightlessness → distilled spirit → measurement fermentation index

Process conditions:

Soaking conditions, impregnate 20min by 60-70 DEG C；Liquefaction condition: 85-90 DEG C, Thermostable α-Amylase, liquefaction is added 90min；55-60 DEG C, carbohydrase is added in saccharification condition, and be saccharified 20min.

Ingredient: corn flour 60g, water 130mL, Thermostable α-Amylase 2 × 10⁴U/mL, 30 μ L, carbohydrase 1 × 10⁵U/mL, 90 μ L, 7.5 × 10²U/mL acid protease 1.2mL: nutritive salt 1mL (MgSO₄150g/L、KH₂PO₄75g/L, urea 81g/L, Filtering, 4 DEG C of preservations)；

One ring yeast cells of picking, equipped in the test tube of 5mL primary-seed medium, 30 DEG C of stationary cultures are pressed for access 10% inoculum concentration is inoculated into the triangular flask equipped with 45mL secondary seed medium, after 30 DEG C of stationary culture 16h to logarithmic phase Phase is inoculated into corn thick mash fermentation culture medium, 30 DEG C of standing for fermentation by 10% inoculum concentration.It is primary every 12h weighing, it is lost when twice It is less than 1g, fermentation ends again.Thick mash 100mL, water 100mL are taken after fermentation, steam 100mL wine sample.Measure weightlessness (i.e. CO₂ Accumulate discharge amount), the fermenting properties index such as alcoholic strength and residual reducing sugar, as a result such as table 3, the alcohol after recombinant bacterial strain fermentation contains Amount and residual sugar content no significant difference compared with starting strain, the gene knockout in this example will not be to the basic fermenting property of bacterial strain It adversely affects.

The fermenting property of 3 recombinant bacterial strain H α of table, 5 (500) Δ H and starting strain α 5 measures

Note: shown data are the average value of three parallel laboratory test results.

Gas chromatograph: Agilent 7890c；Chromatographic column: white wine dedicated columns, AT.LZP-930,230 DEG C, the μ of 50m × 320 m×1μm；Detector: fid detector, detector temperature: 200 DEG C；Carrier gas: High Purity Nitrogen, flow velocity 5mL/min；Testing conditions: journey Sequence heating, 50 DEG C of holdings 8min, 5 DEG C/min are raised to 120 DEG C, keep 8min；Injector temperature: 200 DEG C；Sample volume: 1.0 μ L；Point Stream mode: it shunts, split ratio 10:1；As a result such as table 4. the results show that isobutanol in the higher alcohol of 5 (500) Δ H of recombinant bacterial strain H α Content reaches 42.24mg/L, reduces 47.85% than starting strain α 5；Isoamyl alcohol content reaches 123.96mg/L, than going out bacterium germination Strain α 5 has dropped 23.14%.

The fermentation index (unit mg/mL) of 4 recombinant bacterial strain H α of table, 5 (500) Δ H and starting strain α 5

Note: shown data are the average value of three parallel test results.

Sequence table

<110>University Of Science and Technology Of Tianjin

<120>a kind of seamless gene knockout method of efficient saccharomyces cerevisiae and its application

<160> 14

<170> SIPOSequenceListing 1.0

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<210> 2

<211> 45

<212> DNA

<213>primer yh1.0 (SphI-RUnknown)

<400> 2

gtgccaagct tgcatgcggg gtaccattaa gggttctcga gagct 45

<210> 3

<211> 28

<212> DNA

<213>primer BAT2-A (KpnI-FUnknown)

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ggggtacccg ctcctttcca aacatctt 28

<210> 4

<211> 40

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<213>primer BAt2-a-r (Unknown)

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ccacgagttt taagaacgat agtatcgcta ttgctacgta 40

<210> 5

<211> 40

<212> DNA

<213>primer BAT2-b-F (Unknown)

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aagatgtttg gaaaggagcg gagctcgttt tcgacactgg 40

<210> 6

<211> 26

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<210> 9

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<212> DNA

<213>primer BAT2-B-FR (Unknown)

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ccacgagttt taagaacgat tagggataac agggtaattg gatggacgca aagaag 56

<210> 10

<211> 18

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<400> 10

ttcactggga ccctttca 18

<210> 11

<211> 22

<212> DNA

<213>primer AHB-A-D (Unknown)

<400> 11

gcttctaatc cgtacttcaa ta 22

<210> 12

<211> 18

<212> DNA

<213>primer AHB-B-U (Unknown)

<400> 12

atgcgtcaat cgtatgtg 18

<210> 13

<211> 18

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<213>primer AHB-B-D (Unknown)

<400> 13

tgaaatcctt gtccagct 18

<210> 14

<211> 1131

<212> DNA

<213>saccharomyces cerevisiae (Saccharomyces cerevisiae)

<400> 14

atgaccttgg cacccctaga cgcctccaaa gttaagataa ctaccacaca acatgcatct 60

aagccaaaac cgaacagtga gttagtgttt ggcaagagct tcacggacca catgttaact 120

gcggaatgga cagctgaaaa agggtggggt accccagaga ttaaacctta tcaaaatctg 180

tctttagacc cttccgcggt ggttttccat tatgcttttg agctattcga agggatgaag 240

gcttacagaa cggtggacaa caaaattaca atgtttcgtc cagatatgaa tatgaagcgc 300

atgaataagt ctgctcagag aatctgtttg ccaacgttcg acccagaaga gttgattacc 360

ctaattggga aactgatcca gcaagataag tgcttagttc ctgaaggaaa aggttactct 420

ttatatatca ggcctacatt aatcggcact acggccggtt taggggtttc cacgcctgat 480

agagccttgc tatatgtcat ttgctgccct gtgggtcctt attacaaaac tggatttaag 540

gcggtcagac tggaagccac tgattatgcc acaagagctt ggccaggagg ctgtggtgac 600

aagaaactag gtgcaaacta cgccccctgc gtcctgccac aattgcaagc tgcttcaagg 660

ggttaccaac aaaatttatg gctatttggt ccaaataaca acattactga agtcggcacc 720

atgaatgctt ttttcgtgtt taaagatagt aaaacgggca agaaggaact agttactgct 780

ccactagacg gtaccatttt ggaaggtgtt actagggatt ccattttaaa tcttgctaaa 840

gaaagactcg aaccaagtga atggaccatt agtgaacgct acttcactat aggcgaagtt 900

actgagagat ccaagaacgg tgaactactt gaagcctttg gttctggtac tgctgcgatt 960

gtttctccca ttaaggaaat cggctggaaa ggcgaacaaa ttaatattcc gttgttgccc 1020

ggcgaacaaa ccggtccatt ggccaaagaa gttgcacaat ggattaatgg aatccaatat 1080

ggcgagactg agcatggcaa ttggtcaagg gttgttactg atttgaactg a 1131

Claims (6)

1. a kind of seamless gene knockout method of efficient saccharomyces cerevisiae, it is characterised in that: with the monoploid α 5 of saccharomyces cerevisiae AY15 For starting strain, BAT2 gene is target gene, realizes the seamless knockout to gene BAT2.

2. the seamless gene knockout method of efficient saccharomyces cerevisiae according to claim 1, it is characterised in that: steps are as follows:

HERP1.0 segment is amplified with PCR method, which is cloned into YEp352 plasmid, constructs YHERP1.0 plasmid；

Being expanded respectively with PCR method and knocking out target gene BAT2 upstream and downstream length is 500bp and the homologous sequence piece of 683bp Section, then obtains seamless fusion segment by fusion DNA vaccine；

The segment is cloned on plasmid YHERP1.0, is obtained recombinant plasmid YHERP1.0 (500)；

Expand the segment knocked out on target gene upstream 597bp and YHERP1.0 (500) respectively with PCR method；

The two segments are imported in saccharomyces cerevisiae simultaneously by lithium acetate transformation method, are carried out by YPGly+AF screening and culturing medium The screening of first step integration, obtains the mutant strain containing resistant gene；

With 30 DEG C of galactolipin induced medium, 180rpm is cultivated for 24 hours, is coated on after 100 times of dilution containing the fluoro- 2 '-deoxidation urine of 5- On the synthetic media plate of glycosides, 30 DEG C of culture 36h obtain the Wine brewing yeast strain of second step integration recombination.

3. the seamless gene knockout method of efficient saccharomyces cerevisiae according to claim 1, it is characterised in that:

It is induced for 24 hours in 500bp forward direction homologous sequence, the induced medium of 0.5g/100mL galactolipin, it is anti-to dilute 100 times of coatings To under conditions of screening and culturing medium, the probability of second step homologous recombination can be made to reach 6.86 × 10^-4。

4. the seamless gene knockout method of efficient saccharomyces cerevisiae according to claim 1, it is characterised in that:

Liquor fermentation is carried out under conditions of its fermenting property is unaffected, compared with parent strain, normal propyl alcohol, isobutanol and different Amylalcohol content reduces 20.32%, 47.85% and 23.14% respectively, has achieved the purpose that yield of higher alcohol.

5. the seamless gene knockout method of efficient saccharomyces cerevisiae according to claim 1, it is characterised in that:

Whole amino acid in starting strain saccharomyces cerevisiae (Saccharomyces cerevisiae) CICC32315 monoploid α 5 Transaminase encoding gene BAT2 carries out seamless knockout to realize.Specially saccharomyces cerevisiae H α 5 (500) Δ H.

6. the efficient seamless gene knockout method of saccharomyces cerevisiae as described in claim 1 is applied to answering for the genetic modification of yeast With.