CN103232947A - Freezing resistant Saccharomyces cerevisiae bacterial strain and construction method thereof - Google Patents
Freezing resistant Saccharomyces cerevisiae bacterial strain and construction method thereof Download PDFInfo
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Abstract
The invention discloses a freezing resistant Saccharomyces cerevisiae bacterial strain and construction method thereof, concretely is a freezing resistant Saccharomyces cerevisiae BY-14A delta U constructed by a non-trace knock-out gene NTH1, and the preservation number is CGMCC NO.7379. The freezing resistant Saccharomyces cerevisiae constructed by a non-trace gene knock-out method is realized by fusing PCR and a yeast integrative plasmid YIplac211, and knocking out a gene NTH1 for coding neutral trehalase. The invention realizes a rapid and high efficiency non-trace knock out of Saccharomyces cerevisiae NTH1 gene, and intracellular mycose content increases by 134% than that of the parent bacterial strain without influences to other fermentation performances of the bacterial strain BY-14A delta U; after the cells in the liquid dough are frozen for 21 days, the survival rate reaches to 78%, which is 2.6 times of the parent bacterial strain. The genome in the constructed microzyme does not have any residual exogenous genes, therefore the product can be used for industrial production safely.
Description
Technical field
The present invention relates to the genetically engineered field, specifically relate to cold-resistant bread yeast bacterial strain and the seamless knockout technique of gene thereof of freezing of a strain.
Background technology
Bread yeast (Baker ' s Yeast, formal name used at school Saccharomyces cerevisiae) be a kind of important industrial microorganism, containing abundant nutritive ingredient, is most important microbial starter culture, biological raising agent and biological nutritive agent in the wheaten food production processes such as steamed bun, bread, biscuit.Along with developing rapidly of frozen dough production technology, cold-resistantly freeze bread yeast and the cold-resistant mechanism of freezing has obtained broad research, in the developed country such as American-European, Japanese, the frozen dough technology also has been widely used in baking food.The deep freeze resistance of common bread yeast is poor is the principal element of restriction frozen dough technical development.Trehalose is a kind of autoprotection agent in yeast cell, can avoid frostbite at refrigerating process by Cell protection.Increase the freeze-resistant performance that the yeast intracellular trehalose content can improve bread yeast.But along with the concern of the public to food safety, the bacterial strain built by traditional genetic method, due to residual nonself foreign gene, its production application is restricted.Therefore be necessary yeast is carried out to seamless gene knockout, to guarantee can not stay any foreign DNA.
Originally saccharomycetic seamless modification is in order to remove selection markers, in order to carry out the polygene operation in single bacterial strain.Remove selection markers and mainly contain two kinds of methods, a kind of is after target gene knocks out, and utilizes and knocks out the homologous recombination between direct repetitive sequence in element (hisG).Another kind of be the system that knocks out of utilizing recombinase-mediated, such as Cre/Loxp system etc.Utilize first method, at first need to build the plasmid with " hisG-URA3-hisG ", then use the long primer Direct PCR to obtain knocking out element, after knocking out goal gene by conversion, oppositely screening again, utilize the homologous recombination between direct repetitive sequence (hisG) to remove selection markers.In this method, long primer comprises two portions sequence, and a part is and the sequence of plasmid annealing that another part is the gene order identical with the flanking sequence of target gene both sides.The transformant obtained by this method, on its genomic target position, can a residual tumor-necrosis factor glycoproteins.Utilize second method, by transformation and selection mark both sides with recombinase site knock out element in yeast, a step is integrated and to be realized knocking out of goal gene, then transforms the plasmid of another coding recombinase, to realize the removal of resistance marker.In the transformation of industrial yeast, this system has very high efficiency, but it still can stay exogenous array (single loxp site), and is carrying out polygene while knocking out, and these sites that stay have increased the possibility that chromosome rearrangement occurs.2006, Japanese scholars was used the long primer with target gene one side 40bp tumor-necrosis factor glycoproteins, and the seamless element that knocks out built by merging PCR can be realized easily restructuring, and can not stay foreign gene.But, in this method, the tumor-necrosis factor glycoproteins recombination efficiency of 40bp is lower.In a word, this area still needs a kind of efficient seamless gene knockout method.
Summary of the invention
The technical problem that the present invention solves has been to provide cold-resistant bread yeast bacterial strain and the seamless knockout technique of gene thereof of freezing of a strain.
For solving the problems of the technologies described above, the present invention adopts following technical scheme:
Bread yeast bacterial strain provided by the invention is the cold-resistant bread yeast bacterial strain that freezes with quick fermentation performance, is specially bread yeast (Saccharomyces cerevisiae) BY-14a Δ U.This bacterium is preserved in Chinese microbial preservation management committee's common micro-organisms center March 28 in 2013, and (be called for short CGMCC, address is: No. 3, great Tun road, Chaoyang District, BeiJing, China city first), deposit number is CGMCC No.7379.
Described bread yeast bacterial strain is in the impregnable situation of other leavening properties, after fermentation, intracellular trehalose content accounts for dry cell weight 11.7%, than parent strain, has improved 134%, and cell is in liquid dough after freezing 21 days, survival rate reaches 78%, is 2.6 times of parent strain.
Described bread yeast bacterial strain specifically can carry out seamless knocking out by the full sequence of the neutral trehalase encoding gene NTH1 to starting strain bread yeast (Saccharomyces cerevisiae) CICC31616 (Chinese industrial microbial strains preservation administrative center, the public can obtain starting strain CICC31616 by this preservation administrative center) to be realized.
Seamless the knocking out of said gene can be realized with following methods.The related concrete operation method of each step is with reference to existing bibliographical information, as Joseph Sambrook etc., and " molecular cloning experiment guide " second edition, Science Press, 1995.
Obtain sudden change ura3 fragment by PCR method, it imported to the bread yeast starting strain by the Lithium Acetate chemical transformation, by homologous recombination to realize the URA3 transgenation of starting strain.By PCR method increase the respectively upstream and downstream that knocks out target gene NTH1, the homologous sequence fragment that length is 0.5～2.0kb, then, by merging PCR, the upstream and downstream sequence is fused into to seamless fragment.This fragment is cloned into to yeast integrated plasmid YIplac211 upper, acquisition can be integrated the plasmid knocked out.Knock out in the upstream homology arm or downstream homology arm of plasmid in integration, select suitable single restriction enzyme site, by the plasmid enzyme restriction linearizing.By the Lithium Acetate chemical transformation, linearizing integration is knocked out to plasmid and import in bread yeast, with containing the yeast synthetic medium screening generation the first step of uridylic (uracil), not integrating the yeast mutant of restructuring.To integrate the yeast mutant of recombinating through the generation the first step of identifying, through containing the dull and stereotyped oppositely screening of 5-fluororotic acid (5-FOA) synthetic medium, obtain the yeast mutant that the generation second step is integrated restructuring.Obtain the normal URA3 gene fragment of starting strain by PCR method, it is imported in the yeast mutant that second step integration restructuring has occurred, with the ura3 marker gene of reverse mutation.
Bread yeast bacterial strain of the present invention can be applicable in bread production.
The present invention provides a kind of being specifically designed to identify described cold-resistant gene order of freezing the bread yeast bacterial strain simultaneously, this gene order is to take the described cold-resistant fragment that bread yeast strain gene group is template amplification of freezing with NTH1-D-F and NTH1-U-R primer pair, and its sequence is as shown in table 1.
Beneficial effect:
The present invention is directed in bread yeast tradition gene knockout selection markers residual, inconvenience is carried out polygene and is knocked out research, and the problem of residual foreign gene, and a strain bread yeast bacterial strain and a kind of efficient seamless gene knockout method are provided.The present invention not only can be used for studying function and the metabolic mechanism of yeast genes, and due to the not residual any foreign gene of obtained mutant strain, can safety for industrial production.
The accompanying drawing explanation
Fig. 1 is the phenotype proof diagram with sudden change ura3 gene bread yeast bacterial strain BY-14a Δ U.
Fig. 2 is for integrating the structure schematic flow sheet that knocks out plasmid YIplac211-UD.
Fig. 3 is for integrating the electrophoresis proof diagram that knocks out plasmid YIplac211-UD.
Fig. 4 integrates the restructuring schematic flow sheet for integrating two steps that knock out plasmid YIplac211-UD and Yeast genome.
Fig. 5 integrates the electrophoresis proof diagram of the bacterial strain BY-14a Δ U-U of restructuring for the first step occurs.
Fig. 6 integrates the electrophoresis proof diagram of the bacterial strain BY-14a Δ U-Δ N of restructuring for second step occurs.
Fig. 7 is the part sequencer map of the integration recombinant bacterial strain BY-14a Δ N target gene position of reply ura3 sudden change.
Embodiment
Below by specific embodiment narration the present invention.Unless stated otherwise, in the present invention, technique means used is method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, but not limits the scope of the invention, and the spirit and scope of the invention are only limited by claims.To those skilled in the art, under the prerequisite that does not deviate from essence of the present invention and scope, various changes that the material component in these embodiments and consumption are carried out or change and also belong to protection scope of the present invention.
Embodiment 1: the seamless structure that knocks out neutral trehalase gene NTH1 bread yeast
The starting strain CICC31616 that this example is used, in this example, improved bread yeast bacterial strain (Saccharomyces cerevisiae) is preserved in Chinese microorganism strain management committee common micro-organisms center (CGMCC) on March 28th, 2013, and preserving number is CGMCC No.7379.R.D.Gietz is seen in the source of described plasmid YIplac211, and A.Sugino, New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites.Gene 74 (1988) 527-34.Described escherichia coli DH5a is purchased from Takara company.Described YPD substratum is general perfect medium, and described yeast synthetic medium (SD) composition is 2% glucose, 0.67%YNB, not containing the aminoacid mixture solution of uridylic, and solid medium is containing 2% import agar powder.
According to the Yeast genome data in Genebank and integrated plasmid sequence, designed each primer in following embodiment.
Primer used in table 1. the present embodiment
Annotate: underscore means restriction enzyme site, and italic means to merge the overlap of PCR the primer.
(1) with the structure of the bread yeast BY-14a Δ U of defectiveness ura3 gene
Use the Yeast genome of Solarbio company to extract test kit, extract the genome of laboratory strains W303-1a.Utilize primer pair URA3-F and URA3-R, the genome of W303-1a of take is template, pcr amplification bacterial strain W303-1a sudden change ura3 fragment.After test kit reclaims, fragment is imported in standard bread yeast BY-14a by the Lithium Acetate chemical transformation, the homologous recombination by between sudden change ura3 and normal URA3, realize the URA3 transgenation of reference culture.Bacteria suspension after conversion, coat and added on 5-fluororotic acid (5-FOA) and uridylic (uracil) yeast synthetic medium (SD) flat board, cultivates 48h for 30 ℃, obtains the bread yeast BY-14a Δ U with defectiveness ura3 gene.The mutant strain obtained is definite correct through the phenotype checking, and BY-14a Δ U is not long on the yeast synthetic medium as shown in Figure 1, well-grown on the YPD flat board.
(2) integrate the structure that knocks out plasmid YIplac211-UD
At first, the genome of standard bread yeast BY-14a of take is template, uses primer NTH1-SphI and NTH1-LRR to obtain the gene order of 1283bp on the left of target gene NTH1 by PCR; Use the gene order of primer NTH1-RLL and NTH1-BamHI amplified target gene right side 616bp, respectively called after NTH1-D and NTH1-U simultaneously.Then, the mixture of NTH1-D and NTH1-U of take is template, adds primer NTH1-SphI and NTH1-BamHI to be merged PCR, obtains seamless fusion fragment.After cutting glue recovery (test kit), use respectively BamHI and SphI double digestion, finally be subcloned in the corresponding restriction enzyme site of integrated plasmid YIplac211, integration knocks out plasmid YIplac211-UR and successfully constructs, and builds flow process as shown in Figure 2.Fig. 3 is for integrating the electrophoresis proof diagram that knocks out plasmid YIplac211-UD: wherein swimming lane 1 is for setting out plasmid YIplac211 electrophoresis result; Swimming lane 2 is integrated plasmid YIplac211-UD electrophoresis result; Swimming lane 3 is result after BamHI single endonuclease digestion plasmid YIplac211; Swimming lane 4 is result after BamHI single endonuclease digestion integrated plasmid YIplac211-UD; Swimming lane M is 1Kb DNA Ladder maker; Swimming lane 5 is result after BamHI and SphI double digestion integrated plasmid YIplac211-UD.
Above-mentioned fusion PCR method is the primer that employing well known in the art has complementary end, formation has overlap PCR product, by PCR product overlap, extend, thereby the method that any DNA fragment is coupled together, this technology does not need the digestion of restriction endonuclease and the processing of ligase enzyme, just can realize the external connection of DNA fragmentation, this makes integrated plasmid after two steps are integrated restructuring, and Yeast genome can residual external source restriction enzyme site.
(3) bread yeast neutral trehalase gene NTH1's knocks out
The SpeI enzyme is cut integrated plasmid YIplac211-UD; With the Lithium Acetate conversion method, linearizing integrated plasmid YIplac211-UD is imported in bread yeast BY-14a Δ U, after two steps are integrated restructuring, obtain the bread yeast of the seamless NTH1 of knocking out, two steps are integrated regrouping process as Fig. 4.
The first step is integrated the generation of restructuring, is partly to integrate due to the linearization plasmid imported and the homology of Yeast genome, thereby brings whole plasmid into genome.Bacteria suspension after conversion, coat not containing on the yeast synthetic medium flat board of uridylic, cultivates 48h for 30 ℃, obtains the yeast strain BY-14a Δ U-U that the first step is integrated restructuring occurs.The resulting single bacterium colony of random choose, adopting YIp-IN and NTH1-D-OUT is integration site outside primer, NTH1-D-F and NTH1-U-R are the inboard primer of integration site, carry out bacterium colony PCR screening.Fig. 5 integrates the electrophoresis proof diagram of the bacterial strain BY-14a Δ U-U of restructuring for the first step occurs: swimming lane M is DL5000 DNA Ladder maker; Swimming lane 1,2 is respectively and uses outside primer PCR the result, and swimming lane 1 template is BY-14a Δ U, and swimming lane 2 templates are BY-14a Δ U-U; Swimming lane 3,4 is respectively and uses inboard primer PCR the result, and swimming lane 3 templates are BY-14a Δ U, and swimming lane 4 templates are BY-14a Δ U-U.
The restructuring yeast strains BY-14a Δ U-U obtained through screening and identification, get an articulating in 5ml liquid YPD substratum, after 30 ℃ of lower 200rpm vibration 24h, diluting 10 times coats on the yeast synthetic medium flat board that contains 5-fluororotic acid and uridylic, cultivate 48h for 30 ℃, obtain the yeast strain that second step is integrated restructuring occurs.As shown in Figure 4, after the second step recombination and integration, two kinds of results appear: one, if integrate between the tumor-necrosis factor glycoproteins that the upstream homologous sequence forms, yeast reverts back to starting strain BY-14a Δ U; Two, if integrate between the tumor-necrosis factor glycoproteins that the downstream homologous sequence forms, middle all sequences ejects, and realizes that the seamless of NTH1 knocks out, simultaneously not residual any foreign gene on target position.The resulting single bacterium colony of random choose, adopting NTH1-D-F and NTH1-U-R is integration site outside primer, NTH1-IN-F and NTH1-IN-R are the inboard primer of integration site, carry out bacterium colony PCR screening.Fig. 6 integrates the electrophoresis proof diagram of the bacterial strain BY-14a Δ U-Δ N of restructuring for second step occurs: swimming lane M is DL5000 DNA Ladder maker; Swimming lane 1,2 is respectively and uses outside primer PCR the result, and swimming lane 1 template is BY-14a Δ U-Δ N, and swimming lane 2 templates are BY-14a Δ U; Swimming lane 3,4 is respectively and uses inboard primer PCR the result, and swimming lane 3 templates are BY-14a Δ U-Δ N, and swimming lane 4 templates are BY-14a Δ U, and two swimming lane 750bp left and right bands are assorted band.
(4) integrate the reply of recombinant bacterial strain BY-14a Δ U-Δ N sudden change ura3 gene
With (1) described method in this example, utilize primer pair URA3-F and URA3-R, the normal URA3 gene fragment of amplification BY-14a, by chemical conversion, to reply the ura3 gene of BY-14a Δ U-Δ N bacterial strain sudden change, finally construct the not seamless knock-out bacterial strain BY-14a Δ N of residual any exogenous gene sequence.
In order further to verify the sequence situation that knocks out target position, extract the genome of seamless knock-out bacterial strain BY-14a Δ N, use primer NTH1-D-F and NTH1-U-R to carry out pcr amplification, the fragment that obtains 1104bp is sent the order-checking in Hua Da genome company, and result is as Fig. 7.Fig. 7 a is sequence contrast before and after knocking out, and Fig. 7 b is the sequencing result that knocks out rear target position 80bp, and other results do not provide, and can determine that the seamless NTH1 of knocking out successfully constructs.
Embodiment 2: the cold-resistant bread yeast intracellular trehalose that freezes accumulates and the comparison of freezing rear cell survival rate mensuration
Monoploid parent and corresponding recombinant bacterial strain thereof are carried out respectively cultivating in 48 hours simultaneously, and mensuration intracellular trehalose accumulating level and Fluid simulation dough cell survival rate after freezing latter 21 days, the results are shown in Table 2.As shown in the results, after cell cultures 48h, build the bacterial strain intracellular trehalose and account for dry cell weight 11.7%, than parent strain (5.0%), increased by 134%; After the recombinant bacterial strain of monoploid parent and correspondence thereof is carried out respectively cultivating in 48 hours, freezing 21 days, measure cell survival rate, the cell survival rate of mutant strain is 78%, is 2.6 times of starting strain (30%).
The accumulation of table 2 bread bacterial strain intracellular trehalose is measured with freezing rear cell survival rate
Annotate: shown in the data mean value that is three parallel test results.
Claims (5)
1. the cold-resistant bread yeast bacterial strain that freezes of a strain, be specially bread yeast (Saccharomyces cerevisiae) BY-14A Δ U, and deposit number is CGMCC No.7379.
2. the cold-resistant bread yeast bacterial strain that freezes of a strain as claimed in claim 1, is characterized in that, after described bread yeast bacterial strain fermentation, intracellular trehalose content accounts for dry cell weight 11.7%; Cell is in liquid dough after freezing 21 days, and survival rate reaches 78%, is 2.6 times of parent strain.
3. the cold-resistant construction process that freezes the bread yeast bacterial strain of a strain as claimed in claim 1, it is characterized in that, described construction process is by merging two step gene integration methods of round pcr and integrating vector plasmid YIplac211 mediation, knocks out to realize by the gene NTH1 of coding neutral trehalase is seamless.
4. the cold-resistant construction process that freezes the bread yeast bacterial strain of a strain as claimed in claim 3, it is characterized in that, concrete steps comprise: by PCR method, obtain sudden change ura3 fragment, it is imported to the bread yeast starting strain by the Lithium Acetate chemical transformation, realize the URA3 transgenation of starting strain by homologous recombination; Increase respectively and knock out the homologous sequence fragment that target gene NTH1 upstream and downstream, length are 0.5～2.0kb by PCR method, then, by merging PCR, the upstream and downstream sequence is fused into to seamless fragment; This fragment is cloned into to yeast integrated plasmid YIplac211 upper, acquisition can be integrated the plasmid knocked out; Knock out in the upstream homology arm or downstream homology arm of plasmid in integration, select suitable single restriction enzyme site, by the plasmid enzyme restriction linearizing; By the Lithium Acetate chemical transformation, linearizing integration is knocked out to plasmid and import in bread yeast, with containing the yeast synthetic medium screening generation the first step of uridylic, not integrating the yeast mutant of restructuring; To integrate the yeast mutant of recombinating through the generation the first step of identifying, through containing the dull and stereotyped oppositely screening of 5-fluororotic acid synthetic medium, obtain the yeast mutant that the generation second step is integrated restructuring; Obtain the normal URA3 gene fragment of starting strain by PCR method, it is imported in the yeast mutant that two steps integration restructuring have occurred, with the ura3 marker gene of reverse mutation.
5. a strain as claimed in claim 1 or 2 is cold-resistant freezes the application of bread yeast bacterial strain in bread is produced.
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| CN103589748A (en) * | 2013-11-18 | 2014-02-19 | 天津科技大学 | Method for introducing site-specific mutagenesis rapidly and efficiently |
| CN103898140A (en) * | 2014-04-14 | 2014-07-02 | 中国科学院天津工业生物技术研究所 | Simple efficient gene editing method |
| CN104017742A (en) * | 2014-06-20 | 2014-09-03 | 天津科技大学 | High-resistant yeast strain and preparation method thereof |
| CN104131005A (en) * | 2014-08-11 | 2014-11-05 | 天津科技大学 | High-ester-produced saccharomyces cerevisiae strain and method for seamlessly inserting promoter of high-ester-produced saccharomyces cerevisiae strain |
| CN104357471A (en) * | 2014-10-15 | 2015-02-18 | 中国计量学院 | Application of gene knock-out carrier in gene function research of trichoderma brevicompactum |
| CN108676811A (en) * | 2018-05-28 | 2018-10-19 | 郝志敏 | A kind of seamless editor's carrier of gene and its application in organism gene editing |
| CN108779470A (en) * | 2015-12-17 | 2018-11-09 | 赢创德固赛(中国)投资有限公司 | The box gene knocked out for homologous recombination in yeast cells |
| CN114426986A (en) * | 2021-12-07 | 2022-05-03 | 南京师范大学 | URA-Blaster method established in gibberella and based on CRISPR technology integration |
| TWI839924B (en) | 2022-10-31 | 2024-04-21 | 財團法人食品工業發展研究所 | Freeze-thaw resistant yeast, preparation method and application thereof |
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| CN103898140B (en) * | 2014-04-14 | 2017-03-22 | 中国科学院天津工业生物技术研究所 | Simple efficient gene editing method |
| CN103898140A (en) * | 2014-04-14 | 2014-07-02 | 中国科学院天津工业生物技术研究所 | Simple efficient gene editing method |
| CN104017742A (en) * | 2014-06-20 | 2014-09-03 | 天津科技大学 | High-resistant yeast strain and preparation method thereof |
| CN104017742B (en) * | 2014-06-20 | 2017-12-19 | 天津科技大学 | One plant height patience yeast strain and its construction method |
| CN104131005A (en) * | 2014-08-11 | 2014-11-05 | 天津科技大学 | High-ester-produced saccharomyces cerevisiae strain and method for seamlessly inserting promoter of high-ester-produced saccharomyces cerevisiae strain |
| CN104131005B (en) * | 2014-08-11 | 2017-02-22 | 天津科技大学 | High-ester-produced saccharomyces cerevisiae strain and method for seamlessly inserting promoter of high-ester-produced saccharomyces cerevisiae strain |
| CN104357471A (en) * | 2014-10-15 | 2015-02-18 | 中国计量学院 | Application of gene knock-out carrier in gene function research of trichoderma brevicompactum |
| CN108779470A (en) * | 2015-12-17 | 2018-11-09 | 赢创德固赛(中国)投资有限公司 | The box gene knocked out for homologous recombination in yeast cells |
| CN108676811A (en) * | 2018-05-28 | 2018-10-19 | 郝志敏 | A kind of seamless editor's carrier of gene and its application in organism gene editing |
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| TWI839924B (en) | 2022-10-31 | 2024-04-21 | 財團法人食品工業發展研究所 | Freeze-thaw resistant yeast, preparation method and application thereof |
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