CN115960733B - Genetically engineered saccharomycete for assembling large fragment DNA, construction method and application thereof - Google Patents
Genetically engineered saccharomycete for assembling large fragment DNA, construction method and application thereof Download PDFInfo
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Abstract
The invention provides a genetically engineered saccharomycete for assembling large fragment DNA, a construction method and application thereof, and relates to the technical fields of molecular biology and synthetic biology. The invention provides genetically engineered yeasts Synbio-Sc1, synbio-Sc2 and Synbio-Sc3 for large fragment DNA assembly. Experiments prove that the genetically engineered saccharomycete can efficiently assemble large-fragment DNA with the length of up to 60kb, has the assembly efficiency of up to 63.5% -93.8%, greatly improves the assembly efficiency and the upper synthesis limit of the large-fragment DNA, can reduce the production cost, and has important significance for industrial research and production of gene synthesis and synthetic biological DNA construction.
Description
Technical Field
The invention relates to the technical fields of molecular biology and synthetic biology, in particular to a genetically engineered saccharomycete for assembling large-fragment DNA, a construction method and application thereof.
Background
With the development of synthetic biology, the scale of artificial synthesis and design modification is larger and larger, and the traditional gene fragments, single metabolic pathways are expanded to a plurality of metabolic pathways and even complete genome. As research demands develop, DNA assembly techniques face increasing challenges. In recent years, DNA assembly techniques have been developed, and small fragment DNA assembly techniques have been relatively mature and stable. In vitro enzymatic assembly is often used for small fragment DNA assembly, such as overlap extension PCR (OE-PCR) method relying on high-fidelity polymerase, golden Gate method relying on endonuclease and ligase, isothermal homologous recombination (Gibson) method relying on the synergistic action of exonuclease, ligase and DNA polymerase, etc. However, the large-fragment DNA has low in-vitro assembly efficiency and is easy to generate base variation due to the large molecular weight, easy breakage and easy formation of a secondary structure, so that the large-fragment DNA mainly utilizes the recombination capability of microbial cells to carry out in-vivo recombination. Common in vivo assembly hosts are mainly model organisms such as escherichia coli, bacillus subtilis, saccharomyces cerevisiae and the like, wherein the saccharomyces cerevisiae is the first choice for large-fragment DNA assembly due to high bearing capacity and high homologous recombination efficiency. Research shows that factors such as the length, the number of DNA fragments, the size of a recombination arm and the like are obviously and negatively related to the assembly success rate, and the current conventional saccharomyces cerevisiae cannot meet the increasing demands in the field of DNA synthesis. How to further improve the in vivo assembly efficiency and DNA size of Saccharomyces cerevisiae is a problem to be solved at present.
There are two linear DNA assembly modes of nonhomologous ligation (Non-homologous end joining, NHEJ) and homologous recombination (Homologous recombination, HR) in Saccharomyces cerevisiae. The homologous recombination process determines the integrity and fidelity of the final assembly construction of the transferred DNA fragment, and is a key process of in vivo assembly of large-fragment DNA. However, the patent for further improving the assembling ability of large fragment DNA in Saccharomyces cerevisiae by gene editing means is not reported.
Disclosure of Invention
In view of the above, the present invention aims to provide a genetically engineered yeast for large-fragment DNA assembly, which can be used as an assembly platform for large-fragment DNA to assemble large-fragment DNA widely and efficiently.
In order to solve the technical problems, the invention provides the following technical scheme:
the invention provides a genetic engineering microzyme for large fragment DNA assembly, which is obtained by taking saccharomyces cerevisiae as an original strain through gene editing; the gene editing comprises any one of the following steps: 1) Knocking out the Ku gene; 2) Knocking out the Ku gene and inserting an expression frame of the ScRad52-M gene at a knocking-out site; 3) The Ku gene was knocked out and the ScRad52 gene was overexpressed.
Preferably, the Saccharomyces cerevisiae includes, but is not limited to Saccharomyces cerevisiae BY4741; the nucleotide sequence of the Ku gene is shown as SEQ ID NO. 1.
Preferably, the ScRad52-M gene expression cassette comprises a GAP promoter, a ScRad52-M gene and an ADH1 terminator; the nucleotide sequences of the GAP promoter, the ScRad52-M gene, the ADH1 terminator and the ScRad52-M gene expression frame are respectively shown as SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5.
Preferably, the overexpressed ScRad52 gene is specifically:
inserting a GAP promoter in front of the Saccharomyces cerevisiae genome ScRad52 gene; the nucleotide sequence of the GAP promoter is shown as SEQ ID NO. 2.
The invention also provides a construction method of the genetically engineered saccharomycete, which comprises the following steps:
taking saccharomyces cerevisiae as an original strain, knocking out a Ku gene on a genome through a CRISPR/Cas9 system to obtain a genetically engineered yeast strain Synbio-Sc1;
on the basis of a genetically engineered yeast strain Synbio-Sc1, integrating a ScRad52-M gene expression frame into a knockout site in the genetically engineered yeast strain Synbio-Sc1 through a CRISPR/Cas9 system to obtain a genetically engineered yeast strain Synbio-Sc2;
on the basis of a genetically engineered yeast strain Synbio-Sc1, the GAP promoter is integrated into the ScRad52 gene in the genetically engineered yeast strain Synbio-Sc1 by a CRISPR/Cas9 system to obtain the genetically engineered yeast strain Synbio-Sc3.
Preferably, the Ku gene on the genome is knocked out by:
respectively constructing pUC57-Donor-ku70 plasmid and pCas9-sgRNA-ku70 plasmid, jointly converting Donor-ku70 linear DNA fragment and pCas9-sgRNA-ku70 plasmid recovered from pUC57-Donor-ku70 plasmid into Saccharomyces cerevisiae BY4741, centrifuging, incubating, culturing, and performing PCR screening cloning to obtain the genetically engineered yeast strain Synbio-Sc1.
Preferably, the pUC57-Donor-ku70 plasmid is assembled from a Donor-ku70 gene fragment onto a pUC57-Kan vector; the nucleotide sequence of the Donor-ku70 gene fragment is shown as SEQ ID NO. 6;
the pCas9-sgRNA-ku70 plasmid is obtained by assembling a sgRNA-ku70 gene fragment on a CRISPR vector; the nucleotide sequence of the sgRNA-ku70 gene fragment is shown as SEQ ID NO. 7.
Preferably, the ScRad52-M gene expression cassette is integrated into the knockout site in the genetically engineered yeast strain Synbio-Sc1 by the following steps:
respectively constructing pUC57-Donor-Rad52 plasmid and pCas9-sgRNA-ku70 plasmid, jointly converting Donor-Rad52 linear DNA fragment and pCas9-sgRNA-ku70 plasmid recovered from pUC57-Donor-Rad52 plasmid into Saccharomyces cerevisiae BY4741, centrifuging, incubating, culturing, and performing PCR screening cloning to obtain genetically engineered yeast strain Synbio-Sc2;
the pUC57-Donor-Rad52 plasmid is obtained by assembling a Donor-Rad52 gene fragment onto a pUC57-Kan vector; the nucleotide sequence of the Donor-Rad52 gene fragment is shown as SEQ ID NO. 8.
Preferably, the GAP promoter is integrated into the genetically engineered yeast strain Synbio-Sc1 prior to the ScRad52 gene by:
construction of pUC57-Donor-P GAP Plasmid and pCas9-sgRNA-Rad52 plasmid will be described from pUC57-Donor-P GAP Donor-P recovered from plasmid GAP The linear DNA fragment and pCas9-sgRNA-Rad52 plasmid are transformed into a genetically engineered yeast strain Synbio-Sc1 together, and the genetically engineered yeast strain Synbio-Sc1 is subjected to centrifugation, incubation and culture, and then PCR screening cloning is carried out, thus obtaining the baseBecause of engineering yeast strain Synbio-Sc3;
the pUC57-Donor-P GAP The plasmid is formed by Donor-P GAP The gene fragment is assembled on a pUC57-Kan vector; said Donor-P GAP The nucleotide sequence of the gene fragment is shown as SEQ ID NO. 9;
the pCas9-sgRNA-Rad52 plasmid is obtained by assembling a sgRNA-Rad52 gene fragment onto a CRISPSR vector; the nucleotide sequence of the sgRNA-Rad52 gene fragment is shown as SEQ ID NO. 10.
The invention also provides application of the genetically engineered saccharomycete or the construction method in high-efficiency assembly of large-fragment DNA.
The invention has the beneficial effects that:
the invention modifies Saccharomyces cerevisiae genome by CRISPR/Cas9 method to obtain three large segment DNA high-efficiency assembled genetically engineered yeasts Synbio-Sc1, synbio-Sc2 and Synbio-Sc3. Experiments prove that the genetically engineered saccharomycete can efficiently assemble large-fragment DNA with the length of up to 60kb, and the assembly efficiency is up to 63.5% -93.8%. The invention greatly improves the assembly efficiency and the upper limit of synthesis of large-fragment DNA, can reduce the production cost, and has important significance for industrial research and production of gene synthesis and synthesis biological construction.
Drawings
FIG. 1 is a genetic map of a Cas9 plasmid in a yeast genome editing system; wherein a is pCas9-sgRNA-ku70 plasmid and b is pCas9-sgRNA-Rad52 plasmid.
FIG. 2 is a genetic map of the Donor plasmid in the yeast genome editing system; wherein a is pUC57-Donor-ku70 plasmid, b is pUC57-Donor-Rad52 plasmid, and c is pUC57-Donor-P GAP A plasmid.
FIG. 3 shows the result of electrophoresis of PCR verification of clones after assembly of large fragment DNA of different sizes; wherein a, b, c and d are the PCR electrophoresis results of the assembly of Plasmid-20 BY strains BY4741, sc1, sc2 and Sc3, e, f, g and h are the PCR electrophoresis results of the assembly of Plasmid-30 BY strains BY4741, sc1, sc2 and Sc3, and i, j, k and l are the PCR electrophoresis results of the assembly of Plasmid-60 BY strains BY4741, sc1, sc2 and Sc3.
FIG. 4 shows the assembly efficiency of different yeasts for large DNA fragments of different sizes; wherein a is the efficiency of transformation of yeast with respect to DNA of 20kb in size, b is the efficiency of transformation of yeast with respect to DNA of 30kb in size, and c is the efficiency of transformation of yeast with respect to DNA of 60kb in size.
Detailed Description
The invention provides a genetic engineering microzyme for large fragment DNA assembly, which is obtained by taking saccharomyces cerevisiae as an original strain through gene editing; the gene editing comprises any one of the following steps: 1) Knocking out the Ku gene; 2) Knocking out the Ku gene and inserting an expression frame of the ScRad52-M gene at a knocking-out site; 3) The Ku gene was knocked out and the ScRad52 gene was overexpressed.
In the present invention, the Saccharomyces cerevisiae preferably includes Saccharomyces cerevisiae BY4741 but is not limited to BY4741. In the invention, the nucleotide sequence of the Ku gene is shown as SEQ ID NO. 1; the ScRad52-M gene is preferably obtained BY codon optimization of a RAD52 gene derived from Saccharomyces cerevisiae BY4741; the nucleotide sequence of the ScRad52-M gene is shown as SEQ ID NO. 3; the nucleotide sequence of the ScRad52-M gene expression frame is shown as SEQ ID NO. 5.
In the invention, the over-expression ScRad52 gene specifically comprises: inserting a GAP promoter in front of the Saccharomyces cerevisiae genome ScRad52 gene; the nucleotide sequence of the GAP promoter is shown as SEQ ID NO. 2.
The invention also provides a construction method of the genetically engineered saccharomycete, which comprises the following steps:
taking saccharomyces cerevisiae as an original strain, knocking out a Ku gene on a genome through a CRISPR/Cas9 system to obtain a genetically engineered yeast strain Synbio-Sc1;
on the basis of a genetically engineered yeast strain Synbio-Sc1, integrating a ScRad52-M gene expression frame into a knockout site in the genetically engineered yeast strain Synbio-Sc1 through a CRISPR/Cas9 system to obtain a genetically engineered yeast strain Synbio-Sc2;
on the basis of a genetically engineered yeast strain Synbio-Sc1, the GAP promoter is integrated into a genome ScRad52 gene by a CRISPR/Cas9 system to obtain the genetically engineered yeast strain Synbio-Sc3.
In the present invention, it is preferable to knock out Ku gene on genome by: respectively constructing pUC57-Donor-ku70 plasmid and pCas9-sgRNA-ku70 plasmid, jointly converting Donor-ku70 linear DNA fragment and pCas9-sgRNA-ku70 plasmid recovered from pUC57-Donor-ku70 plasmid into Saccharomyces cerevisiae BY4741, centrifuging, incubating, culturing, and performing PCR screening cloning to obtain the genetically engineered yeast strain Synbio-Sc1. In the invention, the pUC57-Donor-ku70 plasmid is obtained by assembling a Donor-ku70 gene fragment onto a pUC57-Kan vector; the nucleotide sequence of the Donor-ku70 gene fragment is shown as SEQ ID NO. 6; the pCas9-sgRNA-ku70 plasmid is obtained by assembling a sgRNA-ku70 gene fragment on a CRISPR vector; the nucleotide sequence of the sgRNA-ku70 gene fragment is shown as SEQ ID NO. 7.
In the present invention, it is preferable to integrate the expression cassette of the ScRad52-M gene into the knockout site in the genetically engineered yeast strain Synbio-Sc1 by: respectively constructing pUC57-Donor-Rad52 plasmid and pCas9-sgRNA-ku70 plasmid, jointly converting Donor-Rad52 linear DNA fragment and pCas9-sgRNA-ku70 plasmid recovered from pUC57-Donor-Rad52 plasmid into Saccharomyces cerevisiae BY4741, centrifuging, incubating, culturing, and performing PCR screening cloning to obtain genetically engineered yeast strain Synbio-Sc2. In the invention, the pUC57-Donor-Rad52 plasmid is obtained by assembling a Donor-Rad52 gene fragment onto a pUC57-Kan vector; the nucleotide sequence of the Donor-Rad52 gene fragment is shown as SEQ ID NO. 8.
In the present invention, it is preferable that the GAP promoter is integrated into the ScRad52 gene in the genetically engineered strain Synbio-Sc1 by the following steps: construction of pUC57-Donor-P GAP Plasmid and pCas9-sgRNA-Rad52 plasmid will be described from pUC57-Donor-P GAP Donor-P recovered from plasmid GAP The linear DNA fragment and pCas9-sgRNA-Rad52 plasmid are transformed into a genetically engineered yeast strain Synbio-Sc1 together, and the genetically engineered yeast strain Synbio-Sc3 can be obtained by carrying out PCR screening cloning after centrifugation, incubation and culture. In the present invention, the pUC57-Donor-P GAP The plasmid is formed by Donor-P GAP The gene fragment is assembled on a pUC57-Kan vector; said Donor-P GAP Fragments of genesThe nucleotide sequence SEQ ID NO. 9; the pCas9-sgRNA-Rad52 plasmid is obtained by assembling a sgRNA-Rad52 gene fragment onto a CRISPSR vector; the nucleotide sequence of the sgRNA-Rad52 gene fragment is shown as SEQ ID NO. 10.
The invention provides application of the genetically engineered saccharomycete or the construction method in high-efficiency assembly of large-fragment DNA.
In the invention, experimental reagents such as a bacterial culture medium, a yeast auxotroph culture medium, a plasmid extraction reagent, a PCR product purification and agarose gel recovery reagent are all produced and prepared by Suzhou Hongsu biotechnology Co. The used endonucleases and ligases and Gibson kits are purchased from commercial sources; the primers and plasmid vectors required by the experiment are produced and constructed by the Songhong biotechnology Co., ltd. In Suzhou, and the construction modes are Gibsonassembly recombination.
The present invention will be described in detail below with reference to examples for the purpose of making the objects, technical solutions and advantages of the present invention more apparent, but they should not be construed as limiting the scope of the present invention.
In the following examples, conventional methods are used unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
EXAMPLE 1 Gene synthesis and construction of the target plasmid
1. Gene synthesis
All of the following gene sequences were synthesized by hong Suzhou Biotech Co., ltd:
1) GAP promoter (SEQ ID NO. 2)
2)ScRad52-M(SEQ ID NO.3)
3) ADH1 terminator (SEQ ID NO. 4)
4) ScRad52-M expression cassette (SEQ ID NO. 5)
5)Donor-ku70(SEQ ID NO.6)
6)Donor-Rad52(SEQ ID NO.8)
7)Donor-P GAP (SEQ ID NO.9)
8)sgRNA-ku70(SEQ ID NO.7)
9)sgRNA-Rad52(SEQ ID NO.10)
2. Construction of the target plasmid
The sgRNA-ku70 and sgRNA-Rad52 were assembled onto CRISPR vectors by Gibson homologous recombination, and the remaining gene fragments were all assembled onto pUC57-Kan vectors, the plasmid details of which are shown in Table 1 below.
TABLE 1 plasmids used in the construction of strains
EXAMPLE 2 construction of genetically modified Strain
1. Construction of the Strain Synbio-Sc1
The preparation method of the saccharomyces cerevisiae competent cells comprises the following steps:
(1) Inoculating Saccharomyces cerevisiae BY4741 to a His auxotroph solid culture medium (SD-His), and culturing at 30 ℃ for 48 hours to obtain obvious colonies;
(2) Selecting a monoclonal and inoculating the monoclonal into a fresh YPD liquid culture medium, and culturing at a constant temperature of 30 ℃ for 12 hours;
(3) Inoculating the bacterial liquid to a fresh YPD liquid culture medium according to the ratio of 1:10, and culturing at a constant temperature of 30 ℃ until the OD value of the bacterial liquid reaches 0.9-1.0;
(4) Centrifuging the bacterial liquid at 4000rpm and 4 ℃, collecting yeast cells, and carefully removing the supernatant;
(5) Resuspending the yeast cells with pre-chilled sterile ultrapure water, centrifuging at 4000rpm at 4 ℃ to collect the yeast cells, repeating 2 times;
(6) The yeast cells were resuspended using 0.1M LiAc solution, 1/10 volume of 1M DTT was added and incubated at 30℃for 1h;
(7) Centrifuging the bacterial liquid at 4000rpm and 4 ℃, collecting yeast cells, and carefully removing the supernatant;
(8) The yeast cells were resuspended using a pre-chilled 1M sorbitol solution, collected by centrifugation at 4000rpm at 4℃and repeated 2 times;
(9) The yeast cells were resuspended using a small amount of pre-chilled 1M sorbitol solution, ice-bath for 1h;
(10) And subpackaging the yeast competent cells, and preserving at a low temperature of-80 ℃.
The pUC57-Donor-ku70 plasmid was digested, and a Donor-ku70 linear DNA fragment was recovered. The pCas9-sgRNA-ku70 plasmid and the Donor-ku70 linear DNA fragment were transformed into yeast competent cells using PEG/LiAc chemistry at 500ng and 1000ng, respectively. After yeast transformation, competent cells were collected by centrifugation and inoculated into fresh medium (YPD mixed with 1M sorbitol solution in equal ratio) for incubation for 4h, and yeast cells were collected by centrifugation again and plated onto His amino acid-deficient (SD-His) solid medium. After 2-3 days, single colony is selected and inoculated into fresh SD-His liquid culture medium for culture, after 2 days, the bacteria are centrifuged and resuspended by using a proper amount of sterile water, PCR primer YKU-SEQF/-SEQR is designed on the periphery of the homology arm at the upstream and downstream of the knockout site, the PCR is used for screening clone, and the PCR product is recovered for sequencing verification. BY4741 DeltaKu strain was finally obtained and designated as strain Synbio-Sc1.
2. Construction of the Strain Synbio-Sc2
The preparation method of the saccharomyces cerevisiae competent cells is the same as that of the step 1.
The pUC57-Donor-Rad52 plasmid was digested, and a Donor-Rad52 linear DNA fragment was recovered. The pCas9-sgRNA-ku70 plasmid and the Donor-Rad52 linear DNA fragment were transformed into yeast competent cells using PEG/LiAc chemistry at 500ng and 1000ng, respectively. After yeast transformation, competent cells were collected by centrifugation and inoculated into fresh medium (YPD mixed with 1M sorbitol solution in equal ratio) for incubation for 4h, and yeast cells were collected by centrifugation again and plated onto His amino acid-deficient (SD-His) solid medium. After 2-3 days, single colony is selected and inoculated into fresh SD-His liquid culture medium for culture, after 2 days, the culture medium is centrifuged and appropriate amount of sterile water is used for resuspending thalli, primers Rad 52-JF 1/Rad52-JJR2 are designed on the periphery of homologous arms on the upstream and downstream of the knocked-in genes, PCR is used for screening the clone, and PCR products are recovered for sequencing verification. BY4741 DeltaKu is finally obtained: : the ScRad52-M strain is designated as strain Synbio-Sc2.
3. Construction of the Strain Synbio-Sc3
The preparation method of the saccharomyces cerevisiae competent cells is the same as that of the step 1.
Cleavage of pUC57-Donor-P GAP Plasmid and recovery of Donor-P GAP Linear DNA fragments. pCas9-sgRNA-Rad52 plasmid and Donor-P using PEG/LiAc chemistry GAP The linear DNA fragment was transformed into yeast competent cells at 500ng and 1000ng, respectively. After yeast transformation, competent cells were collected by centrifugation and inoculated into fresh medium (YPD mixed with 1M sorbitol solution in equal ratio) for incubation for 4h, and yeast cells were collected by centrifugation again and plated onto His amino acid-deficient (SD-His) solid medium. Picking single colony after 2-3 days, inoculating to fresh SD-His liquid culture medium for culturing, centrifuging after 2 days, re-suspending thallus with proper amount of sterile water, designing primer P around homologous arm at the upstream and downstream of knock-in gene GAP -SEQF/P GAP SEQR, screening clones using PCR, and recovering PCR products for sequencing verification. BY4741 Delta Ku Chromosome Site213984 is finally obtained: : p (P) GAP The strain was designated as strain Synbio-Sc3.
Wherein the PCR amplification primer sequences of the different genetically engineered yeast strains are shown in Table 2.
TABLE 2 PCR amplification primers for genetically engineered yeast strains
Example 3 verification of assembly efficiency for different assembly platforms
In order to further test the assembly efficiency of 3 genetically engineered yeasts prepared in example 2 as large DNA fragments in different sizes, three large DNA fragments of 20kb, 30kb and 60kb were selected for assembly test, and the final constructions were designated as Plasmid-20, plasmid-30 and Plasmid-60, respectively.
Competent cells were prepared with BY4741, synbio-Sc1, synbio-Sc2, synbio-Sc3 strains, respectively, and the procedure was as in step 1 of example 2.
Finally, three DNAs of Plasmid-20, plasmid-30 and Plasmid-60 were split into 5, 7 and 10 small fragments (4 kb to 6 kb), respectively, and synthesized in vitro and assembled onto pUC57 vector in advance. The correct monoclonal shake bacteria were selected and the individual minifragment plasmids of Plasmid-20, plasmid-30 and Plasmid-60 were prepared. The DNA fragments were digested and recovered as materials for the final construction of the assembly test.
And (3) jointly converting the assembly materials of Plasmid-20, plasmid-30 and Plasmid-60 obtained in the last step, namely small-size DNA fragments, into yeast competent cells by a PEG/LiAc chemical method so as to test the assembly capacity of different yeast engineering strains serving as large-fragment DNA fragment assembly platforms. The amount of each small DNA fragment added was 1000ng, and after yeast transformation, competent cells were collected by centrifugation and inoculated into fresh medium (YPD mixed with 1M sorbitol solution in equal ratio) for incubation for 4h, and yeast cells were collected by centrifugation again and plated onto His amino acid-deficient solid medium. After 2-3 days, 96 single colonies from the corresponding plates of each yeast strain are respectively picked and respectively inoculated into fresh SD-His liquid medium for culture, after 2 days, the bacteria are centrifuged and resuspended by using a proper amount of sterile water for PCR identification of the positive rate of the clones.
PCR identification primers were designed based on the periphery of the homologous recombination arms between small fragments, and 3 pairs of primers (as shown in Table 3) were designed for each final construct, and clonality identification was performed using a multiplex PCR method. Theoretically, if the final construction assembly is successful, the PCR electrophoresis result will show a 3-entry band.
TABLE 3 PCR identification primers for large fragment DNA assembly
Based on the PCR electrophoresis results shown in FIG. 3, the ratios of bands of 0 (None), 1 (One band), 2 (Two bands) and 3 (Three bands) in all clones were calculated, respectively. Clones in which 3-item bands were all expanded were regarded as correct clones, and the assembly efficiency was calculated therefrom, and experimental results were obtained as shown in table 4 and fig. 4.
TABLE 4 Assembly results of large fragment DNA in different genetically engineered yeasts
It can be seen that the assembly efficiency of BY4741 is 10.4% -53.1%, the assembly efficiency of Synbio-Sc1 is 21.9% -63.5%, the assembly efficiency of Synbio-Sc2 is 42.7% -93.8%, and the assembly efficiency of Synbio-Sc3 is 46.9% -91.7%. And the larger the size of the DNA fragment, the lower the assembly efficiency of the assembly platform. Compared with BY4741, the three assembly platforms of Synbio-Sc1, synbio-Sc2 and Synbio-Sc3 have significantly improved assembly efficiency for large DNA fragments of different sizes. And the improvement range of the assembly efficiency of Synbio-Sc2 and Synbio-Sc3 is better than that of Synbio-Sc1, and no obvious difference exists between the Synbio-Sc2 and the Synbio-Sc3. The result shows that the recombination capability of 3 genetically engineered yeast strains is obviously enhanced, and the assembly efficiency and the upper assembly limit of large-size DNA fragments are obviously improved.
As can be seen from the above examples, the genetically engineered yeast strain of the present invention can be used as a platform for assembling large-fragment DNA, and can be widely and efficiently used for assembling large-fragment DNA such as complete metabolic pathway, thereby greatly promoting the efficiency and upper limit of gene synthesis.
The foregoing description is only illustrative of the present invention and is not intended to limit the scope of the invention, and all equivalent structures or equivalent processes or direct or indirect application in other related arts are included in the scope of the present invention.
Claims (9)
1. The application of the genetically engineered saccharomycete in high-efficiency assembly of large-fragment DNA is characterized in that the genetically engineered saccharomycete is obtained by taking saccharomyces cerevisiae as an original strain through gene editing; the gene editing comprises any one of the following steps: 1) Knocking out the Ku gene and inserting an expression frame of the ScRad52-M gene at a knocking-out site; 2) The Ku gene was knocked out and the ScRad52 gene was overexpressed.
2. The use according to claim 1, wherein the saccharomyces cerevisiae includes, but is not limited to saccharomyces cerevisiae BY4741; the nucleotide sequence of the Ku gene is shown as SEQ ID NO. 1.
3. The use according to claim 1, characterized in that the expression cassette of the ScRad52-M gene comprises the GAP promoter, the ScRad52-M gene and the ADH1 terminator; the nucleotide sequences of the GAP promoter, the ScRad52-M gene, the ADH1 terminator and the ScRad52-M gene expression frame are respectively shown as SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5.
4. The use according to claim 1, wherein the overexpressed ScRad52 gene is specifically:
inserting a GAP promoter in front of the Saccharomyces cerevisiae genome ScRad52 gene; the nucleotide sequence of the GAP promoter is shown as SEQ ID NO. 2.
5. The use according to claim 1, wherein the method for constructing genetically engineered yeasts comprises the steps of:
taking saccharomyces cerevisiae as an original strain, knocking out a Ku gene on a genome through a CRISPR/Cas9 system to obtain a genetically engineered yeast strain Synbio-Sc1;
on the basis of a genetically engineered yeast strain Synbio-Sc1, integrating a ScRad52-M gene expression frame into a knockout site in the genetically engineered yeast strain Synbio-Sc1 through a CRISPR/Cas9 system to obtain a genetically engineered yeast strain Synbio-Sc2;
on the basis of a genetically engineered yeast strain Synbio-Sc1, the GAP promoter is integrated into the ScRad52 gene in the genetically engineered yeast strain Synbio-Sc1 by a CRISPR/Cas9 system to obtain the genetically engineered yeast strain Synbio-Sc3.
6. The use of claim 5, wherein the Ku gene on the genome is knocked out by:
respectively constructing pUC57-Donor-ku70 plasmid and pCas9-sgRNA-ku70 plasmid, jointly converting Donor-ku70 linear DNA fragment and pCas9-sgRNA-ku70 plasmid recovered from pUC57-Donor-ku70 plasmid into Saccharomyces cerevisiae BY4741, centrifuging, incubating, culturing, and performing PCR screening cloning to obtain the genetically engineered yeast strain Synbio-Sc1.
7. The use according to claim 6, wherein said pUC57-Donor-ku70 plasmid is obtained by assembling a Donor-ku70 gene fragment onto a pUC57-Kan vector; the nucleotide sequence of the Donor-ku70 gene fragment is shown as SEQ ID NO. 6;
the pCas9-sgRNA-ku70 plasmid is obtained by assembling a sgRNA-ku70 gene fragment on a CRISPR vector; the nucleotide sequence of the sgRNA-ku70 gene fragment is shown as SEQ ID NO. 7.
8. Use according to claim 5, characterized in that the expression cassette of the ScRad52-M gene is integrated into the knockout site in the genetically engineered yeast strain Synbio-Sc1 by the following steps:
respectively constructing pUC57-Donor-Rad52 plasmid and pCas9-sgRNA-ku70 plasmid, jointly converting Donor-Rad52 linear DNA fragment and pCas9-sgRNA-ku70 plasmid recovered from pUC57-Donor-Rad52 plasmid into Saccharomyces cerevisiae BY4741, centrifuging, incubating, culturing, and performing PCR screening cloning to obtain genetically engineered yeast strain Synbio-Sc2;
the pUC57-Donor-Rad52 plasmid is obtained by assembling a Donor-Rad52 gene fragment onto a pUC57-Kan vector; the nucleotide sequence of the Donor-Rad52 gene fragment is shown as SEQ ID NO. 8.
9. The use according to claim 5, characterized in that the GAP promoter is integrated into the genetically engineered yeast strain Synbio-Sc1 before the ScRad52 gene by:
respectively constructing pUC57-Donor-PGAP plasmid and pCas9-sgRNA-Rad52 plasmid, jointly converting Donor-PGAP linear DNA fragment and pCas9-sgRNA-Rad52 plasmid recovered from pUC57-Donor-PGAP plasmid into genetically engineered yeast strain Synbio-Sc1, centrifuging, incubating, culturing, and performing PCR screening cloning to obtain genetically engineered yeast strain Synbio-Sc3;
the pUC57-Donor-PGAP plasmid is obtained by assembling a Donor-PGAP gene fragment on a pUC57-Kan vector; the nucleotide sequence of the Donor-PGAP gene fragment is shown as SEQ ID NO. 9;
the pCas9-sgRNA-Rad52 plasmid is obtained by assembling a sgRNA-Rad52 gene fragment onto a CRISPSR vector; the nucleotide sequence of the sgRNA-Rad52 gene fragment is shown as SEQ ID NO. 10.
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