CN109136254B

CN109136254B - Efficient saccharomyces cerevisiae traceless gene knockout method and application thereof

Info

Publication number: CN109136254B
Application number: CN201810748064.8A
Authority: CN
Inventors: 张翠英; 肖冬光; 李凭; 王建辉; 郭学武; 陈叶福; 董健; 杜丽平; 马立娟; 于爱群
Original assignee: Tianjin University of Science and Technology
Current assignee: Tianjin University of Science and Technology
Priority date: 2018-07-10
Filing date: 2018-07-10
Publication date: 2021-06-04
Anticipated expiration: 2038-07-10
Also published as: CN109136254A

Abstract

The invention discloses a method for efficiently knocking out a traceless gene in saccharomyces cerevisiae, wherein a forward homologous sequence, galactose concentration and galactose induction time in a traceless gene knocking-out system are optimized, and the probability of homologous recombination in the second step reaches 6.86 multiplied by 10^‑4. By taking haploid alpha 5 of saccharomyces cerevisiae AY15 as an initial strain and BAT2 as a target gene, efficient traceless knockout of wild saccharomyces cerevisiae strain gene BAT2 is realized, and through a white spirit fermentation experiment, the n-propanol, isobutanol and isoamyl alcohol content of modified strains are respectively reduced by 20.32%, 47.85% and 23.14% compared with that of parent strains, so that the aim of low-yield high-grade alcohol is fulfilled. The method can be widely applied to the gene modification of yeast and other microorganisms, and the obtained mutant strain does not have any residual exogenous gene, so that the method can be safely used for industrial production, and provides a beneficial reference for directly knocking out genes in industrial strains.

Description

Efficient saccharomyces cerevisiae traceless gene knockout method and application thereof

The technical field is as follows:

the invention relates to the field of genetic engineering, in particular to a high-efficiency traceless gene knockout method constructed in saccharomyces cerevisiae and a saccharomyces cerevisiae strain with low high alcohol yield constructed by the method.

Background art:

saccharomyces cerevisiae is a typical eukaryotic cell model and important model organism, and its properties include: the characteristics of fast growth and reproduction, short metabolic cycle, easy separation and culture and the like make the saccharomyces cerevisiae more convenient for genetic engineering and genetic research, and the saccharomyces cerevisiae is called as escherichia coli in eukaryotes. With the continuous development and updating of molecular biology and genetic engineering technology, the breeding method of saccharomyces cerevisiae has been developed from initial natural breeding to modern targeted genetic engineering breeding. Gene knock-out (gene knock-out) is a genetic engineering technology developed in the late 80 th 20 th century, and is a technology for deleting or inactivating specific genes of an organism by a proper method so as to achieve the purpose of modifying a certain gene on a chromosome in a site-specific manner. However, with public concern about food safety, strains constructed by conventional genetic methods have limited production applications due to the residual foreign genes that are not themselves. Therefore, knocking out a specific gene of an organism by using a traceless knockout technology becomes a development direction of genetic breeding at present.

The traceless modification of yeast was initially to remove selection markers for multigene manipulation in a single strain. There are two main methods for removing the selection marker, one is to use a recombinase-mediated knockout system, such as Cre/Loxp system, and Cre recombinase can mediate specific recombination between two LoxP sites to delete the gene between the two sites. Integrating the fragment with two LoxP sites in the same direction and a selection marker on a genome through first chemical transformation; then, a plasmid carrying a gene encoding Cre recombinase is introduced by a second chemical transformation, and all sequences between the two sites are excised by the expression of Cre recombinase, thereby completing the removal of the selection marker. In 2004, Iwaki et al applied this system to yeast, achieving multiple gene knock-out of yeast. The Cre/loxP method has high efficiency, but leaves a foreign sequence (LoxP site) on the chromosome, and when multiple gene knockout is performed, the remaining LoxP site increases the possibility of chromosomal rearrangement. Another method is to use homologous recombination between forward repeat sequences (hisG) in the knockout element, firstly construct a plasmid with 'hisG-URA 3-hisG', then use long primer PCR to directly obtain the knockout element, after transforming and knocking out the target gene, then reverse screening, and use homologous recombination between forward repeat sequences (hisG) to remove the screening marker. In 2012, Dong et al applied this method to achieve traceless knockout of yeast genes, but the probability of this method in inducing the second step of homologous recombination was random, and more than one transformant was produced, so that the probability of obtaining ideal transformants finally was low. In the beginning of 2013, a brand-new artificial endonuclease clustered regulated short palindromic repeats (CRISPR)/CRISPR-associated (Cas)) appears, and the artificial endonuclease is mainly formed by modifying an acquired immune system based on bacteria, and has the characteristics of simple manufacture, low cost and high effect. In 2014, Bao et al firstly utilized a CRISPR-Cas system to realize multigene one-step knockout on Saccharomyces cerevisiae. However, guide rna (sg rna) designed in the system can be mismatched with a non-target DNA sequence when guiding the Cas protein to recognize with the genome, and an unexpected gene mutation, i.e., off-target effect, is introduced, which severely restricts the wide application of the technology. In conclusion, there is still a need in the art for a highly efficient traceless gene knockout method.

The high-grade alcohol is an important metabolite in the alcohol fermentation process of saccharomyces cerevisiae, is also a main flavor substance in alcoholic beverages such as white spirit, wine and the like, the content of the high-grade alcohol and the proportion of various alcohols have important influence on the flavor of the wine, and the proper amount of the high-grade alcohol and the coordinated component ratio can endow the wine with special fragrance, and simultaneously set off the fragrance of ester, so that the taste of the wine is coordinated and soft. The high alcohol content can cause the wine to generate foreign flavor, which affects the flavor and quality of the wine, and the high alcohol has lower oxidation speed in human body than ethanol, and the toxic action on human body is far higher than that of ethanol. Therefore, controlling the content of higher alcohols is an important indicator in modern brewing processes. In the alcohol fermentation process of saccharomyces cerevisiae, there are two higher alcohol metabolic pathways, which are the sugar metabolic synthesis pathway (Harris pathway) and the amino acid catabolism pathway (Ehrlich pathway), respectively. In the amino acid catabolic pathway, the first step in the catabolism of saccharomyces cerevisiae branched chain amino acids is transamination. Research reports have shown that the branched-chain amino acid transaminase encoded by the BAT2 gene plays a very important role in the production of higher alcohols, particularly isobutanol and isoamyl alcohol. Therefore, in order to reduce the amount of higher alcohols produced during fermentation, it would be an effective way to knock out the gene BAT2 encoding amino acid transaminase, which has a significant effect on higher alcohols.

Therefore, the invention constructs a high-efficiency traceless knockout method in saccharomyces cerevisiae by taking the amino acid transaminase coding gene BAT2 as a target gene, can be widely applied to the gene modification of yeast and other microorganisms, and provides a beneficial reference for directly knocking out genes in industrial strains. The constructed low-yield higher alcohol mutant strain can be safely used for industrial production and meets the requirements of related fields of saccharomyces cerevisiae application on yeast.

The invention content is as follows:

the invention aims to provide a high-efficiency saccharomyces cerevisiae traceless gene knockout method and construct a saccharomyces cerevisiae strain with low high alcohol yield by using the method.

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

a high-efficiency saccharomyces cerevisiae traceless gene knockout method takes haploid alpha 5 of saccharomyces cerevisiae AY15 as an initial strain and BAT2 gene as a target gene to realize traceless knockout of gene BAT 2.

The low-yield higher alcohol yeast strain constructed by the invention is realized by carrying out traceless knockout on a complete amino acid transaminase coding gene BAT2 in a CICC32315 haploid alpha 5 of a starting strain Saccharomyces cerevisiae (Saccharomyces cerevisiae). In particular to saccharomyces cerevisiae H alpha 5(500) delta H.

The BAT2 Gene has Gene ID: 853613, the nucleotide sequence is shown in the table.

The above-described traceless knockout of a gene can be achieved by the following method. The specific procedures involved in each step are described in the literature, e.g., Joseph Sambrook et al, molecular cloning, A laboratory Manual, second edition, science Press, 1995.

Amplifying a HERP1.0 fragment by using a PCR method, cloning the fragment to a YEp352 plasmid, and constructing a YHERP1.0 plasmid; respectively amplifying homologous sequence fragments with the upstream and downstream lengths of 500bp and 683bp of the knockout target gene BAT2 by using a PCR method, and then obtaining a seamless fusion fragment by fusion PCR; cloning the fragment to a plasmid YHERP1.0 to obtain a recombinant plasmid YHERP1.0 (500); respectively amplifying and knocking out fragments on 597bp at the upstream of a target gene and YHERP1.0(500) by using a PCR method; introducing the two fragments into saccharomyces cerevisiae simultaneously by a lithium acetate conversion method, and carrying out first-step integration screening on the two fragments by a YPGly + AF screening culture medium to obtain a mutant strain containing a resistance gene; and (3) using galactose to induce the culture medium to culture for 24h at 30 ℃ and 180rpm, diluting by 100 times, coating the diluted product on a synthetic culture medium plate containing 5-fluoro-2' -deoxyuridine, and culturing for 36h at 30 ℃ to obtain the second step of integrated recombinant saccharomyces cerevisiae strains.

Under the condition that the growth performance and the fermentation performance of the saccharomyces cerevisiae are not influenced, the probability of the second integration, recombination, knockout, screening and marking process is 6.86 multiplied by 10^-4。

The saccharomyces cerevisiae strain (H alpha 5(500) delta H) can be used for producing white spirit.

The invention also provides optimization of the induction culture medium, and researches the influence of the content of galactose in the induction culture medium on the second step integration and recombination probability as shown in table 1, wherein the operation process is as follows:

1. the total content of galactose in the induction medium is 0.1g/100mL, 0.5g/100mL, 1g/100mL, 2g/100mL, 3g/100mL, 4g/100mL, 5g/100mL eight gradients, peptone 2g/100mL, yeast extract 1g/100 mL.

2. The mutant strain H alpha 5(500) is subjected to induction culture for 24H by using a galactose medium. After 100-fold dilution, plates of synthetic medium containing 5-fluoro-2' -deoxyuridine were plated.

3. TABLE 1 probability of second step integrative recombination

The invention has the following positive effects:

1. the invention provides a high-efficiency saccharomyces cerevisiae traceless gene knockout method, which overcomes the difficulty that the screening marker residue is remained and the multi-gene knockout is inconvenient to carry out in the traditional gene knockout. Meanwhile, the invention not only can be used for researching the function and metabolic mechanism of the yeast gene, but also can be safely used for industrial production because the obtained mutant strain does not have any exogenous gene left.

2. On the premise of keeping good fermentation performance, the saccharomyces cerevisiae with low-yield high-grade alcohol completely knocks out an amino acid transaminase coding gene BAT2, and through a corn thick mash liquor fermentation experiment, the n-propanol, isobutanol and isoamyl alcohol content of the modified strain is obviously reduced, so that the aim of low-yield high-grade alcohol is fulfilled, and a theoretical basis is laid for brewing alcohol beverage with excellent flavor and more favorable for health.

Description of the drawings:

FIG. 1 is a schematic diagram of the construction process of recombinant plasmid YHERP1.0 (500).

FIG. 2 shows the construction and validation electrophoretograms of recombinant plasmids YHERP1.0 and YHERP1.0 (500).

FIG. 3 is a schematic diagram of a two-step integration recombination process in a traceless knock-out procedure.

FIG. 4 is an electrophoresis chart of the strain H.alpha.5 (500) in which the first step of integration and recombination has occurred.

FIG. 5 shows the second step of electrophoretic verification of the integrated recombinant strain H α 5 (500). DELTA.H.

FIG. 6 is a partial sequencing alignment chart of the target gene position of the integrated recombinant strain H alpha 5(500) delta H, wherein FIG. 6a is a sequence alignment before and after knockout, and FIG. 6b is a sequencing result of the target position 40bp after knockout.

FIG. 7 is a graph of H α 5(500) Δ H vs. α 5 growth.

The specific implementation mode is as follows:

the invention is described below by means of specific embodiments. Unless otherwise specified, all technical means used in the experiments are methods well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention.

Example 1

Construction of traceless knockout amino acid transaminase coding gene BAT2 Saccharomyces cerevisiae

The starting strain used in this example was CICC32315 haploid α 5. (the fragment HERP1.0 is derived from William G.Alexander, Drew T.Doering and Chris Todd Hittinger, High-Efficiency Genome Editing and Allle retrieval in Protopathy and Wild Strains of Saccharomyces genetics, Vol.198, 859-866 November 2014). The E.coli DH 5. alpha. was purchased from Takara. The YPD culture medium is a general complete culture medium; the components of the screening culture medium YPGly + AF are 5% of glycerol, 2% of peptone, 1% of yeast extract powder, 200mg/mL of aminomethyl folic acid, 5mg/mL of sulfanilamide, 5 mu g/mL of thymine and 50 mu g/mL of hypoxanthine; the components of the yeast synthetic medium (SC) are 2% of glucose, 0.17% of YNB, 0.5% of ammonium sulfate and a complete amino acid mixture solution, and the solid medium contains 2% of imported agar powder.

Based on the yeast genome data and YEp352 plasmid sequence in Genebank, each primer in the following examples was designed.

TABLE 2 primers used in this example

Note: underlined and italicized indicate the cleavage sites, italicized indicates the overlapping sequences of the primers used for the fusion PCR, and underlined indicates the cleavage sites added.

TABLE 3 PCR amplification System used in this example

TABLE 4 fusion PCR System used in this example

(1) Construction of plasmid YHERP1.0

The construction process of the plasmid is shown in FIG. 1;

first, the genome of YHWA245 was extracted using a yeast genome extraction kit from Solarbio corporation. PCR amplification of HERP1.0 fragment in strain YHWA245 using primers YH1.0(EcoRI) -F and YH1.0(SphI) -R and using the genome of YHWA245 as template, and recovery with kit; carrying out double enzyme digestion on plasmid YEp352 by using EcoRI and SphI endonucleases, cutting gel and recovering enzyme digestion plasmids; using Clone from Vazyme

II One Step Cloning Kit the digested plasmid was ligated with HERP1.0 fragment and transformed into DH 5. alpha. competent plasmid YHERP 1.0.

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

The construction of the recombinant plasmid YHERP1.0(500) is shown in FIG. 1

Firstly, a yeast genome extraction kit of Solarbio company is used for extracting a haploid alpha 5 genome of CICC 32315. Amplifying 500bp of the upstream of BAT2 gene by using primers BAT2-A (KpnI) -F and BAT 2-A-R; meanwhile, primers BAT2-B-F and BAT2-B (KpnI) -R are used for amplifying 683bp downstream of BAT2 gene, and BAT2-U and BAT2-D are named respectively. Then, a mixture of BAT2-U and BAT2-D was used as a template, and primers BAT2-A (KpnI) -F and BAT2-B (KpnI) -R were added to perform fusion PCR, to obtain a seamless fusion fragment, which was named BAT 2-UD. After gel cutting and recovery (kit), enzyme digestion is carried out by KpnI, subcloning is carried out to the corresponding enzyme digestion site of plasmid YHERP1.0, primers AHB-B-U and BAT2-B (KpnI) -R are used for verifying the positive and negative of single enzyme digestion connection, and the construction of the recombinant plasmid YHERP1.0(500) is successful.

FIG. 2 shows the construction and confirmation electrophoretograms of recombinant plasmids YHHERP1.0 and YHHERP1.0 (500): lane 1 shows the result of electrophoresis of HERP1.0(3159 bp); lane 2 shows the result of electrophoresis of YEp352 plasmid (5100bp) digested with EcoRI and SphI; lane 3 shows the result of plasmid electrophoresis for YHERP 1.0: lane 4 shows the result of electrophoresis of YEp352 plasmid; lane 5 shows the result of electrophoresis of plasmid YHERP1.0 (8300bp) digested with KpnI; lane 6 shows BAT2-U₁Electrophoresis result of D fragment (1183 bp); lane 7 shows the result of plasmid electrophoresis in YHERP1.0 (500); lane 8 shows the result of YHERP1.0(500) single-enzyme ligation verified forward/reverse (1260bp) electrophoresis; lane 9 shows the result of electrophoresis of YHERP1.0(500) plasmid (3800bp and 5700bp) digested with EcoRI; lane M is 15000DNAladder maker.

The fusion PCR method is a method known in the art in which PCR products having overlapping sequences are formed using primers having complementary ends, and arbitrary DNA fragments are ligated by extending the overlapping sequences of the PCR products, and in vitro ligation of DNAs is performed without digestion with an endonuclease or treatment with a ligase.

(3) Knockout of amino acid transaminase encoding gene (BAT2) in Saccharomyces cerevisiae

Amplifying the 597bp on the left side of BAT2 by using primers BAT2-A-F and BAT2-A-RF to obtain a 635bp length segment, and naming BAT 2-A-SF; meanwhile, primers BAT2-B-FR and BAT2-B (KpnI) -R are used for amplifying on YHERP1.0(500) to obtain a fragment with the length of about 4700bp, which is named as RS-HERP 1.0-BAT 2-UD; and (3) introducing the fragments BAT 2-A-SF and RS-HERP 1.0-BAT 2-UD into the saccharomyces cerevisiae strain alpha 5 by using a lithium acetate conversion method, and performing two-step integration and recombination to obtain the saccharomyces cerevisiae with trace-free knockout of BAT2, wherein the two-step recombination process is shown in figure 3.

The first integration step occurs because the two fragments introduced are integrated with homologous portions of the yeast genome, and the homologous portions between the two fragments are also integrated, thereby integrating the entire fragment into the α 5 genome. The transformed suspension was plated on YPGly + AF selection medium plates and cultured at 30 ℃ for 7d to obtain yeast strain H.alpha.5 (500) in which the first step of whole recombination occurred. Randomly selecting the obtained single colony, and performing colony PCR screening by using AHB-A-U and AHB-A-D as upstream verification primers of the integration site and AHB-B-U and AHB-B-D as downstream verification primers of the integration site. FIG. 4 shows the electrophoretic verification of the strain H.alpha.5 (500) in which the first step of integration and recombination has occurred: lane M is DL5000 DNA Ladder maker; lanes 1 and 2 were verified by PCR using the forward verification primer, respectively, with the template in lane 1 being H α 5(500) and the template in lane 2 being α 5; lanes 3 and 4 were verified by PCR using the downstream verification primers, respectively, the template in lane 3 is H.alpha.5 (500) and the template in lane 4 is. alpha.5.

And (2) selecting and identifying the obtained recombinant saccharomyces cerevisiae strain H alpha 5(500), connecting a loop to 5mL of galactose induction culture medium, culturing at 30 ℃, 180rpm for 24H, diluting by 100 times, coating on a synthetic culture medium plate containing 5-fluoro-2' -deoxyuridine, and culturing at 30 ℃ for 36H to obtain the second step of integrated recombinant saccharomyces cerevisiae strain. Randomly selecting the obtained single colony, performing colony PCR screening by adopting AHB-A-U and AHB-B-D primers, and verifying that the probability of second integration and recombination can reach 6.86 multiplied by 10^-4The method can quickly and efficiently recover the screening marker, is convenient to reuse, and achieves the aim of traceless knockout. FIG. 5 shows the electrophoretic patterns of the strain H.alpha.5 (500). DELTA.H in which the second integration and recombination step has occurred: lane M is DL5000 Ladder maker; lane 1 shows the results of PCR using the primers AHB-A-U and AHB-B-D, and the template in Lane 1 is H.alpha.5 (500). DELTA.H.

To further verify the sequence condition of the knockout target position, the genome of the traceless knockout strain H alpha 5(500) delta H is extracted, PCR amplification is carried out by using primers AHB-A-U and AHB-B-D, a 2605bp fragment is obtained and sent to Huada gene company for sequencing, and the sequencing result is shown in FIG. 6. FIG. 6a is sequence comparison before and after knockout, FIG. 6b is sequencing result of target position 40bp after knockout, other results are not given, and it can be determined that construction of traceless knockout gene BAT2 Saccharomyces cerevisiae strain is successful.

Example 2

Traceless knockout BAT2 Saccharomyces cerevisiae strain H alpha 5(500) determination of delta H and alpha 5 growth curves

Selecting single colonies of H alpha 5(500) delta H and alpha 5, inoculating the single colonies into 50mL YEPD liquid culture medium, carrying out shaking culture at 30 ℃ and 180rpm for 24H, inoculating the enlarged culture bacterial liquid into 3 bottles of 50mLYEPD culture medium according to the inoculum size of 1:100, carrying out shaking culture under the same condition, respectively taking 0.5mL of bacterial culture suspension after 0H, 1H, 2H, 3H, 4H, 5H, 6H, 7H, 8H, 9H, 10H, 11H, 12H, 13H, 14H and 15H after culture, centrifuging for 1min at 10000rpm, and then re-suspending bacterial sludge by using deionized water. Correcting zero point of 7200 type visible spectrophotometer with deionized water, and performing colorimetric determination on OD of bacterial liquid at wavelength of 600nm₆₀₀The value is obtained. Absorbance value (OD) of bacterial liquid at each time point₆₀₀) The growth curves of the strains are plotted on the ordinate and the culture time on the abscissa, the growth curves are shown in FIG. 7, and it can be seen from the growth curves that the growth performances of H alpha 5(500) delta H and the alpha 5 of the starting strain are not different.

Example 3

Corn thick mash fermentation experiment of traceless knockout BAT2 saccharomyces cerevisiae strain H alpha 5(500) delta H and alpha 5

Respectively and simultaneously carrying out a corn thick mash fermentation experiment on the recombinant strain H alpha 5(500) delta H and the starting strain alpha 5, wherein a route diagram of a fermentation process is as follows:

soaking corn flour → liquefying → saccharifying → adding bacteria and fermenting → weighing weightlessness → distilling alcohol → measuring fermentation index

The process conditions are as follows:

soaking at 60-70 deg.C for 20 min; liquefaction conditions: adding high temperature resistant alpha-amylase at 85-90 deg.C, and liquefying for 90 min; saccharifying at 55-60 deg.C for 20 min.

Preparing materials: corn flour 60g, water 130mL, high temperature resistant alpha-amylase 2X 10⁴U/mL, 30. mu.L, glucoamylase 1X 10⁵U/mL，90μL，7.5×10²U/mL acidic protease 1.2 mL: nutritive salt 1mL (MgSO)₄ 150g/L、KH₂PO₄75g/L and 81g/L of urea, filtering and storing at 4 ℃;

selecting a torula yeast cell, inoculating into a test tube containing 5mL of first-level seed culture medium, standing at 30 deg.C for culture, inoculating into the culture container according to 10% inoculation amountIn a triangular flask with 45mL of secondary seed culture medium, standing and culturing for 16h at 30 ℃ to the later stage of logarithmic phase, inoculating the corn thick mash fermentation culture medium according to the inoculum size of 10%, and standing and fermenting at 30 ℃. Weighing once every 12h, and finishing fermentation when the weight loss of two times is less than 1 g. After the fermentation is finished, 100mL of thick mash and 100mL of water are taken, and 100mL of wine sample is distilled out. Determination of weight loss (i.e. CO)₂Cumulative discharge amount), alcohol content, residual reducing sugar and other fermentation performance indexes, and the results are shown in table 3, the alcohol content and the residual sugar content of the recombinant strain after fermentation are not obviously different from those of the original strain, and the gene knockout in the embodiment does not have adverse effect on the basic fermentation performance of the strain.

TABLE 3 fermentation Performance determination of recombinant Strain H α 5(500) Δ H and starting Strain α 5

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

Gas chromatograph: agilent 7890 c; a chromatographic column: a column special for white spirit, AT.LZP-930, 230 ℃, 50m multiplied by 320 mu m multiplied by 1 mu m; a detector: FID detector, detector temperature: 200 ℃; carrier gas: high-purity nitrogen with the flow rate of 5 mL/min; detection conditions are as follows: heating at 50 deg.C for 8min, heating at 5 deg.C/min to 120 deg.C, and maintaining for 8 min; sample introduction temperature: 200 ℃; sample introduction amount: 1.0 μ L; a flow splitting mode: splitting, wherein the splitting ratio is 10: 1; the results are shown in Table 4, the result shows that the content of isobutanol in the higher alcohol of the recombinant strain H alpha 5(500) delta H reaches 42.24mg/L, and is reduced by 47.85 percent compared with the original strain alpha 5; the content of isoamyl alcohol reaches 123.96mg/L, which is reduced by 23.14 percent compared with the alpha 5 of the original strain.

TABLE 4 fermentation index (in mg/mL) for recombinant strain H α 5(500) Δ H and starting strain α 5

Note: the data shown are the average of the results of three replicates.

Sequence listing

<110> Tianjin science and technology university

<120> high-efficiency saccharomyces cerevisiae traceless gene knockout method and application thereof

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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

1. A saccharomyces cerevisiae traceless gene knockout method is characterized in that: taking haploid alpha 5 of saccharomyces cerevisiae AY15 with the preservation number of CICC32315 as an original strain,BAT2the gene is a target gene, and the pair of genes is realizedBAT2The traceless knockout is carried out; the method comprises the following steps:

using yeast YHWA245 genome as a template, amplifying HERP1.0 fragment by using a PCR method, cloning the fragment into YEp352 plasmid, and constructing YHERP1.0 plasmid; the HERP1.0 fragment is P_GAL1-SCE1-P_TEF1-HSV-TK-T_TEF1；

Respectively amplifying homologous sequence fragments of 500bp upstream and 683bp downstream of the knocked-out target gene BAT2 by using a PCR (polymerase chain reaction) method, and then obtaining a seamless fusion fragment BAT2U-BAT2D by fusion PCR;

cloning the fragment to a plasmid YHERP1.0 to obtain a recombinant plasmid YHERP1.0-BAT2U-BAT 2D;

respectively amplifying fragments of 597bp upstream of the knockout target gene and YHERP1.0-BAT2U-BAT2D by using a PCR method;

simultaneously introducing the two fragments into an original strain by a lithium acetate conversion method, and carrying out first-step integration screening on a YPGly + AF screening culture medium to obtain a mutant strain containing a resistance gene;

culturing with galactose induction culture medium at 30 deg.C and 180rpm for 24h, diluting with 100 times, spreading on synthetic culture medium plate containing 5-fluoro-2' -deoxyuridine, and culturing at 30 deg.C for 36h to obtain second integrated recombinant Saccharomyces cerevisiae strain;

the YPGly + AF culture medium comprises 5% of glycerol, 2% of peptone, 1% of yeast extract powder, 200mg/mL of aminomethyl folic acid, 5mg/mL of sulfanilamide, 5 mu g/mL of thymine and 50 mu g/mL of hypoxanthine;

the content of galactose in the galactose induction medium is 0.5g/100mL, the content of peptone is 2g/100mL, and the content of yeast extract powder is 1g/100 mL;

the upstream and downstream primers for amplifying the HERP1.0 gene segment are respectively as follows: SEQ ID number 1, SEQ ID number 2;

the upstream and downstream primers for amplifying 500bp upstream of BAT2 gene are respectively: SEQ ID number 3, SEQ ID number 4;

the upstream and downstream primers for amplifying 683bp downstream of BAT2 gene are respectively: SEQ ID NO 5, SEQ ID number 6;

the upper and lower primers for amplifying 597bp at the upstream of the target gene are respectively as follows: SEQ ID number 7, SEQ ID number 8;

the upstream and downstream primers for YHERP1.0-BAT2U-BAT2D amplification were: SEQ ID number 9, SEQ ID number 6.

2. The use of a saccharomyces cerevisiae traceless gene knockout method of claim 1 in yeast gene engineering.