CN115960733A - Genetic engineering yeast for large-fragment DNA assembly, construction method and application thereof - Google Patents
Genetic engineering yeast for large-fragment DNA assembly, construction method and application thereof Download PDFInfo
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Abstract
The invention provides a genetic engineering yeast for large-fragment DNA assembly, a construction method and application thereof, relating to the technical field 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 genetic engineering yeast can efficiently assemble large-fragment DNA of 60kb, the assembly efficiency is up to 63.5-93.8%, the assembly efficiency and the upper limit of synthesis of the large-fragment DNA are greatly improved, the production cost can be reduced, and the genetic engineering yeast has important significance for industrial research and production of gene synthesis and synthetic biology DNA construction.
Description
Technical Field
The invention relates to the technical field of molecular biology and synthetic biology, in particular to a genetic engineering yeast for large-fragment DNA assembly, a construction method and application thereof.
Background
With the development of synthetic biology, the scale of artificial synthesis and design and modification is larger and larger, and the traditional gene fragments and single metabolic pathways are expanded to a plurality of metabolic pathways or even complete genomes. As research needs develop, DNA assembly techniques face increasing challenges. In recent years, DNA assembly technology has been developed, and small-fragment DNA assembly technology has become relatively mature and stable. In many cases, small-fragment DNA is assembled by in vitro enzymatic methods, such as overlap-extension PCR (OE-PCR) which relies on high-fidelity polymerase, golden Gate which relies on endonuclease and ligase, isothermal homologous recombination (Gibson) which relies on the synergistic action of exonuclease, ligase and DNA polymerase, and the like. However, since the large-fragment DNA has a large molecular weight, is easily broken, and easily forms a secondary structure, the efficiency of in vitro assembly is low, and base variation is easily caused, so that the large-fragment DNA is mainly recombined in vivo by using the recombination capability of microbial cells. 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 assembly of large-fragment DNA due to high bearing capacity and high homologous recombination efficiency. Researches show that factors such as the length, the number and the size of a recombination arm of a DNA fragment are obviously and negatively related to the assembly power, and the conventional saccharomyces cerevisiae can not meet the increasing requirements in the field of DNA synthesis at present. How to further improve the in vivo assembly efficiency and DNA size of the saccharomyces cerevisiae is a problem to be solved urgently at present.
In Saccharomyces cerevisiae, there are two linear DNA assembly modes, non-Homologous ligation (NHEJ) and Homologous Recombination (HR). 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, no patent for further improving the ability of large-fragment DNA assembly in Saccharomyces cerevisiae by means of gene editing has been reported.
Disclosure of Invention
In view of the above, the present invention aims to provide a genetically engineered yeast for large-fragment DNA assembly, wherein the genetically engineered yeast after being modified 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 yeast for large-fragment DNA assembly, which is obtained by taking saccharomyces cerevisiae as an original strain and carrying out gene editing; the gene editing comprises any one of the following steps: 1) Knocking out a Ku gene; 2) Knocking out a Ku gene and inserting a ScRad52-M gene expression frame into 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 in 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 expression cassette of the ScRad52-M gene 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 GAP promoter in front of ScRad52 gene of the saccharomyces cerevisiae genome; the nucleotide sequence of the GAP promoter is shown as SEQ ID NO. 2.
The invention also provides a construction method of the genetic engineering yeast, which comprises the following steps:
using saccharomyces cerevisiae as an original strain, knocking out a Ku gene on a genome through a CRISPR/Cas9 system to obtain a genetic engineering yeast strain Synbio-Sc1;
on the basis of the genetically engineered yeast strain Synbio-Sc1, integrating the ScRad52-M gene expression frame to 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 the genetically engineered yeast strain Synbio-Sc1, a GAP promoter is integrated into the genetically engineered yeast strain Synbio-Sc1 before a ScRad52 gene through 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 transforming the Donor-ku70 linear DNA fragment and the pCas9-sgRNA-ku70 plasmid which are recovered from the pUC57-Donor-ku70 plasmid into Saccharomyces cerevisiae BY4741, centrifuging, incubating, culturing, and then carrying out PCR screening cloning to obtain the genetic engineering Synbio-Sc1.
Preferably, the pUC57-Donor-ku70 plasmid is obtained by assembling Donor-ku70 gene fragments onto a pUC57-Kan vector; the nucleotide sequence of the Donor-ku70 gene fragment is shown in SEQ ID NO. 6;
the pCas9-sgRNA-ku70 plasmid is obtained by assembling sgRNA-ku70 gene fragments onto a CRIPSR vector; the nucleotide sequence of the sgRNA-ku70 gene fragment is shown in SEQ ID NO. 7.
Preferably, the expression cassette of the ScRad52-M gene is integrated into the knock-out site of the genetically engineered yeast strain Synbio-Sc1 by:
respectively constructing pUC57-Donor-Rad52 plasmid and pCas9-sgRNA-ku70 plasmid, jointly transforming the Donor-Rad52 linear DNA fragment and the pCas9-sgRNA-ku70 plasmid which are recovered from the pUC57-Donor-Rad52 plasmid into Saccharomyces cerevisiae BY4741, centrifuging, incubating, culturing, and then carrying out PCR screening and cloning to obtain the genetic engineering Synbio-Sc2;
the pUC57-Donor-Rad52 plasmid is obtained by assembling Donor-Rad52 gene fragments on a pUC57-Kan vector; the nucleotide sequence of the Donor-Rad52 gene segment is shown as SEQ ID NO. 8.
Preferably, the GAP promoter is integrated into the genetically engineered yeast strain Synbio-Sc1 in front of the ScRad52 gene by the following steps:
construction of pUC57-Donor-P, respectively GAP Plasmids and pCas9-sgRNA-Rad52 plasmid, from pUC57-Donor-P GAP The recovered Donor-P was recovered from the plasmid GAP The linear DNA fragment and pCas9-sgRNA-Rad52 plasmid are jointly transformed into a genetically engineered yeast strain Synbio-Sc1, and PCR screening cloning is carried out after centrifugation, incubation and culture to obtain a genetically engineered yeast strain Synbio-Sc3;
the pUC57-Donor-P GAP The plasmid is composed of Donor-P GAP Assembling the gene fragment to a pUC57-Kan vector; the 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 sgRNA-Rad52 gene fragments onto a CRIPSR vector; the nucleotide sequence of the sgRNA-Rad52 gene fragment is shown in SEQ ID NO. 10.
The invention also provides the application of the genetic engineering yeast or the construction method in the efficient assembly of large-fragment DNA.
The invention has the beneficial effects that:
the invention obtains three large-fragment DNA high-efficiency assembled genetic engineering yeasts Synbio-Sc1, synbio-Sc2 and Synbio-Sc3 by modifying a Saccharomyces cerevisiae genome through a CRISPR/Cas9 method. Experiments prove that the genetic engineering yeast can efficiently assemble large-fragment DNA 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 also reduce the production cost, and has important significance for industrial research and production of gene synthesis and synthetic biology construction.
Drawings
FIG. 1 is a genetic map of 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 gene map of 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 electrophoresis results of PCR verification of clones after assembling large-fragment DNAs of different sizes; wherein a, b, c and d are respectively the PCR electrophoresis results of strains BY4741, sc1, sc2 and Sc3 assembled into Plasmid-20, e, f, g and h are respectively the PCR electrophoresis results of strains BY4741, sc1, sc2 and Sc3 assembled into Plasmid-30, and i, j, k and l are respectively the PCR electrophoresis results of strains BY4741, sc1, sc2 and Sc3 assembled into Plasmid-60.
FIG. 4 shows the assembly efficiency of different yeasts on large fragment DNA of different sizes; wherein, a is the recombination efficiency of the microzyme to the DNA with the size of 20kb, b is the recombination efficiency of the microzyme to the DNA with the size of 30kb, and c is the recombination efficiency of the microzyme to the DNA with the size of 60 kb.
Detailed Description
The invention provides a genetic engineering yeast for assembling large-fragment DNA, 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 a Ku gene; 2) Knocking out a Ku gene and inserting a ScRad52-M gene expression frame into 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 in 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 expression frame of the ScRad52-M gene is shown as SEQ ID NO. 5.
In the invention, the overexpression ScRad52 gene specifically comprises: inserting GAP promoter in front of ScRad52 gene of the saccharomyces cerevisiae genome; the nucleotide sequence of the GAP promoter is shown as SEQ ID NO. 2.
The invention also provides a construction method of the genetic engineering yeast, which comprises the following steps:
using saccharomyces cerevisiae as an original strain, and 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 the genetically engineered yeast strain Synbio-Sc1, integrating the ScRad52-M gene expression frame to 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 the genetically engineered yeast strain Synbio-Sc1, a GAP promoter is integrated to the front of a genome ScRad52 gene through a CRISPR/Cas9 system to obtain the genetically engineered yeast strain Synbio-Sc3.
In the present invention, it is preferable to knock out the Ku gene on the genome by: respectively constructing pUC57-Donor-ku70 plasmid and pCas9-sgRNA-ku70 plasmid, jointly transforming the Donor-ku70 linear DNA fragment and the pCas9-sgRNA-ku70 plasmid which are recovered from the pUC57-Donor-ku70 plasmid into Saccharomyces cerevisiae BY4741, centrifuging, incubating, culturing, and then carrying out PCR screening cloning to obtain the genetic engineering Synbio-Sc1. In the invention, the pUC57-Donor-ku70 plasmid is obtained by assembling a Donor-ku70 gene fragment on a pUC57-Kan vector; the nucleotide sequence of the Donor-ku70 gene fragment is shown in SEQ ID NO. 6; the pCas9-sgRNA-ku70 plasmid is obtained by assembling sgRNA-ku70 gene fragments onto a CRIPSR vector; the nucleotide sequence of the sgRNA-ku70 gene fragment is shown in SEQ ID NO. 7.
In the present invention, preferably, 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 transforming the Donor-Rad52 linear DNA fragment and pCas9-sgRNA-ku70 plasmid which are recovered from the pUC57-Donor-Rad52 plasmid into Saccharomyces cerevisiae BY4741, centrifuging, incubating, culturing, and then carrying out PCR screening cloning to obtain the genetic engineering Synbio-Sc2. In the invention, the pUC57-Donor-Rad52 plasmid is obtained by assembling Donor-Rad52 gene fragments on a pUC57-Kan vector; the nucleotide sequence of the Donor-Rad52 gene segment is shown as SEQ ID NO. 8.
In the present invention, the GAP promoter is preferably integrated into the genetically engineered strain Synbio-Sc1 before the ScRad52 gene by the following steps: construction of pUC57-Donor-P, respectively GAP Plasmids and pCas9-sgRNA-Rad52 plasmid, from pUC57-Donor-P GAP The recovered Donor-P from the plasmid GAP The linear DNA fragment and pCas9-sgRNA-Rad52 plasmid are jointly transformed into a genetically engineered yeast strain Synbio-Sc1, and PCR screening and cloning are carried out after centrifugation, incubation and culture to obtain the genetically engineered yeast strain Synbio-Sc3. In the present invention, pUC57-Donor-P is used GAP The plasmid is composed of Donor-P GAP Assembling the gene fragment to a pUC57-Kan vector; the 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 CRIPSR vector; the nucleotide sequence of the sgRNA-Rad52 gene fragment is shown in SEQ ID NO. 10.
The invention provides the application of the genetic engineering yeast or the construction method in the efficient assembly of large-fragment DNA.
In the invention, experimental reagents such as a bacteria culture medium, a yeast nutrient deficiency culture medium, a plasmid extraction reagent, a PCR product purification reagent, an agarose gel recovery reagent and the like are all produced and prepared by Suzhou Hongxn biotechnology corporation. The used endonuclease, ligase and Gibson kit are purchased from commercial approaches; the primers and the plasmid vector required by the experiment are produced and constructed by Suzhou Hongxn Biotechnology GmbH, and the construction modes adopt Gibsonassambly.
The present invention will be described in detail with reference to examples for better understanding the objects, technical solutions and advantages of the present invention, but the present invention should not be construed as being limited to the scope of the present invention.
In the following examples, unless otherwise specified, all the methods are conventional.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 Gene Synthesis and construction of a plasmid of interest
1. Gene synthesis
The following gene sequences were synthesized by Suzhou Hongxn Biotechnology GmbH:
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 plasmid of interest
sgRNA-ku70 and sgRNA-Rad52 were assembled into a CRIPSR vector by the Gibson homologous recombination method, and the remaining gene fragments were assembled into a pUC57-Kan vector, and the detailed information of the plasmids is shown in table 1 below.
Table 1 plasmids used in the construction of the strains
Example 2 construction of genetically modified strains
1. Construction of the Strain Synbio-Sc1
The preparation method of the saccharomyces cerevisiae competent cell comprises the following steps:
(1) Inoculating Saccharomyces cerevisiae BY4741 to a His auxotrophic solid culture medium (SD-His), and culturing at constant temperature of 30 ℃ for 48h to obtain an obvious colony;
(2) Selecting a single clone, inoculating the single clone into a fresh YPD liquid culture medium, and culturing at the constant temperature of 30 ℃ for 12 hours;
(3) Inoculating the bacterial liquid to a fresh YPD liquid culture medium according to the proportion of 1;
(4) Centrifuging the bacterial liquid at the temperature of 4000rpm and 4 ℃, collecting yeast cells, and carefully removing supernate;
(5) Resuspending yeast cells by using precooled sterile ultrapure water, centrifugally collecting the yeast cells at 4 ℃ at 4000rpm, and repeating for 2 times;
(6) Resuspending yeast cells by using 0.1M LiAc solution, adding 1/10 volume of 1M DTT, and culturing at constant temperature of 30 ℃ for 1h;
(7) Centrifuging the bacterial liquid at the temperature of 4000rpm and 4 ℃, collecting yeast cells, and carefully removing supernate;
(8) Re-suspending the yeast cells by using a pre-cooled 1M sorbitol solution, centrifuging at 4000rpm and 4 ℃, collecting the yeast cells, and repeating for 2 times;
(9) Resuspending yeast cells with a small amount of pre-cooled 1M sorbitol solution, and ice-cooling for 1h;
(10) Subpackaging yeast competent cells, and storing at-80 ℃ low temperature.
The plasmid pUC57-Donor-ku70 was digested with enzymes, and the 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 by PEG/LiAc chemistry, with the addition amounts of 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 proportion) for incubation for 4h, and yeast cells were collected by centrifugation again and spread on His amino acid deficient type (SD-His) solid medium. And after 2-3 days, selecting a single colony, inoculating the single colony into a fresh SD-His liquid culture medium for culture, centrifuging after 2 days, resuspending the thallus by using a proper amount of sterile water, designing PCR primers YKU 70-SEWF/-SEQR at the periphery of upstream and downstream homology arms of a knockout site, screening and cloning by using PCR, and recovering a PCR product for sequencing verification. Finally, a BY4741 delta Ku strain is obtained and named as a strain Synbio-Sc1.
2. Construction of the Strain Synbio-Sc2
The preparation method of the saccharomyces cerevisiae competent cell is the same as the step 1.
The pUC57-Donor-Rad52 plasmid was digested to recover the Donor-Rad52 linear DNA fragment. The pCas9-sgRNA-ku70 plasmid and the Donor-Rad52 linear DNA fragment were transformed into yeast competent cells by PEG/LiAc chemistry, with the addition amounts of 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 proportion) for incubation for 4h, and yeast cells were collected by centrifugation again and spread to His amino acid deficient (SD-His) solid medium. And after 2-3 days, selecting a single colony, inoculating the single colony into a fresh SD-His liquid culture medium for culture, centrifuging the single colony after 2 days, using a proper amount of sterile water for resuspending the thallus, designing primers Rad52-JJF1/Rad52-JJR2 at the periphery of the upstream and downstream homology arms of the knock-in gene, screening and cloning the single colony by using PCR, and recovering a PCR product for sequencing verification. Finally, BY4741 Δ Ku: : the ScRad52-M strain was named Synbio-Sc2 strain.
3. Construction of the Strain Synbio-Sc3
The preparation method of the saccharomyces cerevisiae competent cell is the same as the step 1.
Restriction enzyme of pUC57-Donor-P GAP Plasmid, recovery of Donor-P GAP A linear DNA fragment. The pCas9-sgRNA-Rad52 plasmid and Donor-P were purified by PEG/LiAc chemistry GAP The linear DNA fragments were transformed into yeast competent cells, with the addition amounts of 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 proportion) for incubation for 4h, and yeast cells were collected by centrifugation again and spread on His amino acid deficient type (SD-His) solid medium. Selecting single colony after 2-3 days, inoculating the single colony into a fresh SD-His liquid culture medium for culture, centrifuging after 2 days, using a proper amount of sterile water to resuspend the thallus, and designing a primer P at the periphery of the upstream and downstream homology arms of the knock-in gene GAP -SEQF/P GAP SEQR, clones were screened using PCR and PCR products recovered for sequencing validation. Finally, BY4741 delta Ku Chromosome Site213984 is obtained: : p GAP The strain is named as Synbio-Sc3.
Wherein, the PCR amplification primer sequences of 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 the 3 genetically engineered yeasts prepared in example 2 as an efficient assembly platform for large-fragment DNA to DNA of different sizes, large DNA fragments of three sizes of 20kb, 30kb and 60kb were selected for assembly tests, and the final constructions thereof were named Plasmid-20, plasmid-30 and Plasmid-60, respectively.
Competent cells were prepared from BY4741, synbio-Sc1, synbio-Sc2 and Synbio-Sc3 strains, respectively, in the same manner as step 1 of example 2.
Finally, three DNAs of Plasmid-20, plasmid-30 and Plasmid-60 are split into 5, 7 and 10 small fragments (4 kb-6 kb), synthesized in vitro in advance and assembled to a pUC57 vector. The correct monoclonals were picked, and small fragment plasmids of Plasmid-20, plasmid-30 and Plasmid-60 were prepared, respectively. And (3) enzyme cutting and recovering the DNA fragment as a material for finally constructing an assembly test.
And (3) jointly transforming small-size DNA fragments which are the assembly materials of the Plasmid-20, the Plasmid-30 and the Plasmid-60 obtained in the last step into yeast competent cells by a PEG/LiAc chemical method so as to test the assembly capability of different yeast engineering strains serving as large-fragment DNA fragment assembly platforms. The addition amount of each small DNA fragment is 1000ng, competent cells are collected by centrifugation after yeast transformation and inoculated into a fresh culture medium (YPD is mixed with 1M sorbitol solution in equal proportion) for incubation for 4h, and yeast cells are collected by centrifugation again and spread to a His amino acid defect type solid culture medium. After 2-3 days, 96 single colonies are respectively picked from the corresponding plates of each yeast strain and respectively inoculated into a fresh SD-His liquid culture medium for culture, and after 2 days, the strains are centrifuged and resuspended by using a proper amount of sterile water for PCR (polymerase chain reaction) identification of the positive rate of the clone seeds.
PCR identification primers were designed based on the periphery of the homologous recombination arms between the small fragments, 3 pairs of primers (as shown in Table 3) were designed for each final construction, and the multiplex PCR method was used for clone identification. Theoretically, if the final construction assembly is successful, the PCR electrophoresis result will show 3 bands.
TABLE 3 PCR identification primers for large fragment DNA assembly
Based on the results of PCR electrophoresis shown in FIG. 3, the ratio of 0 (None), 1 (One band), 2 (Two bands) and 3 (Three bands) bands in all clones was calculated, respectively. The clones with 3 bands were all identified as correct clones, and the assembly efficiency was calculated based on them, and the experimental results are shown in table 4 and fig. 4.
TABLE 4 Assembly results of large fragment DNA in different genetically engineered yeasts
As can be seen, 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 less efficient the assembly of the assembly platform. Compared with BY4741, the assembly efficiency of large DNA fragments with different sizes is remarkably improved BY the three assembly platforms of Synbio-Sc1, synbio-Sc2 and Synbio-Sc3. And the improvement range of the assembly efficiency of the Synbio-Sc2 and the Synbio-Sc3 is better than that of the Synbio-Sc1, and the Synbio-Sc2 and the Synbio-Sc3 have no significant difference. The result shows that the recombination capability of the 3 genetic engineering yeast strains is obviously enhanced through genome modification, and the assembly efficiency and the assembly upper limit of large-size DNA fragments are obviously improved.
The examples show that the genetic engineering yeast strain can be used as an assembly platform of large-fragment DNA, can widely and efficiently assemble large-fragment DNA such as a complete metabolic pathway, and greatly promotes the efficiency and the upper limit of gene synthesis.
The above description is only an embodiment of the present invention, and is not intended to limit the scope of the present invention, and all equivalent structures or equivalent processes performed by the present invention or directly or indirectly applied to other related technical fields are also included in the scope of the present invention.
Claims (10)
1. A genetic engineering yeast for large fragment DNA assembly is characterized in that the genetic engineering yeast is obtained by taking saccharomyces cerevisiae as an original strain and carrying out gene editing; the gene editing comprises any one of the following steps: 1) Knocking out a Ku gene; 2) Knocking out a Ku gene and inserting a ScRad52-M gene expression frame into the knocked-out site; 3) The Ku gene was knocked out and the ScRad52 gene was overexpressed.
2. The genetically engineered yeast of claim 1, wherein the saccharomyces cerevisiae includes but is not limited to saccharomyces cerevisiae BY4741; the nucleotide sequence of the Ku gene is shown in SEQ ID NO. 1.
3. The engineered yeast of claim 1, wherein the ScRad52-M gene expression cassette 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 expression cassette of the ScRad52-M gene are respectively shown as SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5.
4. The genetically engineered yeast of claim 1, wherein the overexpressed ScRad52 gene is specifically:
inserting GAP promoter in front of ScRad52 gene of the saccharomyces cerevisiae genome; the nucleotide sequence of the GAP promoter is shown as SEQ ID NO. 2.
5. The method for constructing genetically engineered yeast of claim 1, comprising the steps of:
using saccharomyces cerevisiae as an original strain, and 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 the genetically engineered yeast strain Synbio-Sc1, integrating the ScRad52-M gene expression frame to 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 the genetically engineered yeast strain Synbio-Sc1, a GAP promoter is integrated into the genetically engineered yeast strain Synbio-Sc1 before a ScRad52 gene through a CRISPR/Cas9 system to obtain the genetically engineered yeast strain Synbio-Sc3.
6. The method of constructing according to 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 transforming the Donor-ku70 linear DNA fragment and the pCas9-sgRNA-ku70 plasmid which are recovered from the pUC57-Donor-ku70 plasmid into Saccharomyces cerevisiae BY4741, centrifuging, incubating, culturing, and then carrying out PCR screening and cloning to obtain the genetic engineering Synbio-Sc1.
7. The construction method according to claim 6, wherein the pUC57-Donor-ku70 plasmid is obtained by assembling Donor-ku70 gene fragments into a pUC57-Kan vector; the nucleotide sequence of the Donor-ku70 gene fragment is shown in SEQ ID NO. 6;
the pCas9-sgRNA-ku70 plasmid is obtained by assembling sgRNA-ku70 gene fragments onto a CRIPSR vector; the nucleotide sequence of the sgRNA-ku70 gene fragment is shown in SEQ ID NO. 7.
8. The construction method according to claim 5, wherein the ScRad52-M gene expression cassette is integrated into the knock-out site of the genetically engineered yeast strain Synbio-Sc1 by the following steps:
respectively constructing pUC57-Donor-Rad52 plasmid and pCas9-sgRNA-ku70 plasmid, jointly transforming the Donor-Rad52 linear DNA fragment and the pCas9-sgRNA-ku70 plasmid which are recovered from the pUC57-Donor-Rad52 plasmid into Saccharomyces cerevisiae BY4741, centrifuging, incubating, culturing, and then carrying out PCR screening and cloning to obtain the genetic engineering Synbio-Sc2;
the pUC57-Donor-Rad52 plasmid is obtained by assembling Donor-Rad52 gene fragments on a pUC57-Kan vector; the nucleotide sequence of the Donor-Rad52 gene segment is shown as SEQ ID NO. 8.
9. The method according to claim 5, wherein the GAP promoter is integrated into the genetically engineered yeast strain Synbio-Sc1 before the ScRad52 gene by the following steps:
construction of pUC57-Donor-P, respectively GAP Plasmids and pCas9-sgRNA-Rad52 plasmid, from pUC57-Donor-P GAP The recovered Donor-P from the plasmid GAP The linear DNA fragment and the pCas9-sgRNA-Rad52 plasmid are jointly transformed into a genetically engineered yeast strain Synbio-Sc1, and PCR screening cloning is carried out after centrifugation, incubation and culture to obtain a genetically engineered yeast strain Synbio-Sc3;
the pUC57-Donor-P GAP The plasmid is composed of Donor-P GAP Assembling the gene fragment to a pUC57-Kan vector; the 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 CRIPSR vector; the nucleotide sequence of the sgRNA-Rad52 gene fragment is shown in SEQ ID NO. 10.
10. The use of the genetically engineered yeast of any one of claims 1-4 or the method of construction of any one of claims 5-9 for efficient assembly of large fragments of DNA.
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