Corynebacterium recombinase screened by blast iteration method and application of corynebacterium recombinase

Abstract

The invention discloses corynebacterium recombinase screened by a blast iteration method and application of the corynebacterium recombinase, and belongs to the technical field of gene editing. At present, no gene editing recombinase with good compatibility exists for non-model strains, especially corynebacterium glutamicum, and the non-model strains are not optimized, so that in order to solve the problem, the invention provides a blast iteration method capable of being used for screening target strain recombinase. A new mode is provided for development of a gene editing tool of a non-model strain, and compared with existing efficient recombinase recET and red systems, the efficiency of the recombinase CauR obtained through screening in the non-model strain corynebacterium glutamicum S9114 is higher.

Description

Technical Field

[0001] The invention relates to a coryneform recombinase screened by a blast iteration method and application thereof, and belongs to the technical field of gene editing. Background Art

[0002] Corynebacterium glutamicum is an amino acid production strain widely used in industrial production. It has been applied to the biosynthesis of high-value products such as amino acids, organic acids and terpenoids. Due to its advantages such as rapid growth, rich variety of metabolites and high yield, Corynebacterium glutamicum has gradually become an ideal host in the field of industrial biosynthesis. Gene editing efficiency is crucial in the construction of microbial cell factories. Using recombinase to improve editing efficiency is an efficient and ideal method.

[0003] In the development of various microbial gene editing tools, current research is mainly focused on model strains, and researchers usually use homologous recombinases to enhance the efficiency of homologous recombination. However, existing recombinase systems, such as RecE / RecT and λRed recombination systems, have limitations in compatibility. Especially in the application of non-model strains, these recombinases have poor compatibility and are difficult to meet the needs of different microbial chassis, resulting in low gene editing efficiency. In addition, many developed gene editing tools are not optimized for non-model strains, which limits their effective application in non-model strains.

[0004] Therefore, although the RecE / RecT recombinase system and the λred system have been widely used in Corynebacterium glutamicum in recent years, it is still necessary to screen efficient recombinases for this strain. This is the key to improving its gene editing efficiency and is also expected to further improve the efficiency and diversity of its metabolite synthesis. Summary of the invention

[0005] In order to solve the problem of gene editing of non-model strain Corynebacterium glutamicum S9114, the present invention provides an iterative Blast method to screen endogenous recombinases with better compatibility to improve its gene editing efficiency. Subsequently, the present invention compares the screened endogenous recombinases of Corynebacterium with the existing efficient recombinases recET and red system. The present invention not only screens out four recombinases that can exert recombination ability in Corynebacterium glutamicum S9114, but also provides a more applicable and efficient solution for genome engineering transformation of non-model microorganisms.

[0006] The first object of the present invention is to provide a gene editing system for Corynebacterium glutamicum, wherein the gene editing system contains a recombinase, and the nucleotide sequence of the recombinase is shown in SEQ ID NO.3.

[0007] In the present invention:

[0008] In order to explore the endogenous recombinase of coryneform bacteria and screen for recombinases with better compatibility with non-model strain Corynebacterium glutamicum S9114, the present invention takes C.glutamicum S9114 as an example, and screens endogenous recombinases in coryneform bacteria by iterative Blast method, and successfully screens multiple recombinases with recT structure, among which the top four recombinases with the highest scores screen recE-like enzymes in the upstream of its genome. Then we further compare the efficiency of the recombinase. First, a basic strain is constructed, in which the ldhA gene is replaced by the Spc resistance gene, and a meaningless sequence is inserted into the Spc gene respectively, for testing the dsDNA recombination ability of the recombinase; the same is true for another basic strain, in which the ldhA gene is replaced by the Spc resistance gene, and the termination codon TAA is used to replace its original 101 amino acids, for testing the recombination ability of ssDNA. By using plasmid pXMJ19 as a vector, tac promoter induced expression of recombinase, and using dsDNA or ssDNA as a template, the recombinase's recombination ability was tested, and finally the CauR recombinase from Corynebacterium aurimucosum ATCC 700975 strain had the highest recombination ability. The present invention successfully screened out a recombinase with efficient recombination function in Corynebacterium glutamicum S9114, and also provided a useful reference for the development of gene editing tools for other non-model strains.

[0009] Furthermore, the Corynebacterium glutamicum includes Corynebacterium glutamicum S9114.

[0010] Furthermore, the gene editing system also contains a dsDNA template and / or an ssDNA template, and the dsDNA template and the ssDNA template are designed according to the target gene to be edited. Specifically, dsDNA is used to achieve gene knockout, and ssDNA is used to achieve single base mutation.

[0011] Furthermore, the recombinase is capable of exerting homologous recombination ability in Corynebacterium glutamicum S9114.

[0012] The second object of the present invention is to provide a gene-editing recombinase of Corynebacterium glutamicum, the nucleotide sequence of the recombinase is shown in SEQ ID NO.3.

[0013] The third object of the present invention is to provide a vector system for gene editing of Corynebacterium glutamicum, wherein the vector system contains an expression vector to which a recombinase with a nucleotide sequence as shown in SEQ ID NO.3 is connected.

[0014] Furthermore, the expression vector includes but is not limited to pXMJ19 plasmid.

[0015] The fourth object of the present invention is to provide a Corynebacterium glutamicum containing a recombinase, the nucleotide sequence of the recombinase being shown in SEQ ID NO.3.

[0016] The fifth object of the present invention is to provide a recombinase-mediated gene editing method for Corynebacterium glutamicum, comprising the step of introducing a recombinase into Corynebacterium glutamicum, wherein the nucleotide sequence of the recombinase is shown in SEQ ID NO.3.

[0017] Furthermore, the method also includes the step of introducing dsDNA and / or ssDNA into Corynebacterium glutamicum.

[0018] Furthermore, the recombinase is expressed episomally via a plasmid.

[0019] Furthermore, the plasmid contains a promoter for initiating the expression of the recombinase; the promoter includes an inducible promoter.

[0020] The sixth object of the present invention is to provide a method for screening a gene editing recombinase for a target strain, comprising the following steps:

[0021] S1. Using the known recombinase as the initial sequence, perform a blast search in the target strain database to obtain the blast result;

[0022] S2. Using all the results of blast in S1 as the initial sequence, repeat step S1 and perform iterative search again until no recombinase with a high score is screened out, and summarize all the identified recombinases. Sequences with scores higher than 400 will be used for the gene editing recombinase of the target strain. Of course, the step of verifying the sequences with scores higher than 400 can also be included.

[0023] Furthermore, the target strain includes Corynebacterium.

[0024] Beneficial effects of the present invention:

[0025] The present invention takes C.glutamicum S9114, a non-model strain, as an example. By iterative blast search method, a series of recT-like recombinases are retrieved from Corynebacterium, and four recombinases are found to have recE structure in the upstream of its genome. Therefore, we tested the recombination efficiency of these four recombinases in detail. The result shows that the recombinase CauR from Corynebacterium aurimucosum ATCC 700975 has good recombination efficiency, and gene editing is performed using the recombinase, which can achieve gene knockout and single base mutation. In addition, compared to existing efficient recombinase recET and red system, the present invention is more efficient in non-model strain Corynebacterium glutamicum S9114, and it can be inferred that the recombinase screened has better compatibility in Corynebacterium glutamicum, and the present invention also provides reference for the rapid development and application of other non-model microbial genetic engineering. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is a schematic diagram of the screening method of the present invention;

[0027] Figure 2 It is the complete screening result of the present invention;

[0028] Figure 3 It is the recombination efficiency test process and results of the present invention;

[0029] Figure 4 It is the sequence comparison result of the recombinase screened in the present invention. DETAILED DESCRIPTION

[0030] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0031] 1. The methods and materials involved in the present invention are as follows:

[0032] (I) Culture medium

[0033] E. coli culture medium LB (g / L): yeast powder 5.0, peptone 10.0, NaCl 10.0

[0034] Seed activation liquid medium BHIS (g / L): brain heart infusion 37.0, sorbitol 91.0.

[0035] Seed activation solid medium BHIS (g / L): brain heart infusion 37.0, sorbitol 91.0, nutrient agar 20.0.

[0036] Competent medium (g / L): peptone 10.0, yeast extract 5.0, NaCl 10.0, glycine 30.0, isoniazid 4.0, and 10 mL of Tween 80.

[0037] After electroporation, the recovery medium BHIS (g / L) was as follows: 5.0% peptone, 2.5% yeast extract, 5.0% NaCl, 18.5% brain heart infusion, and 91.0% sorbitol.

[0038] Solid BHIS medium for transformant coating (g / L): 5.0% peptone, 2.5% yeast extract, 5.0% NaCl, 18.5% brain heart infusion, 91.0% sorbitol, and 20.0% nutrient agar.

[0039] Sterilization conditions: 115°C, 20 min. All culture media used for transformant detection or recombinant bacterial culture were added with 25 mg / L kanamycin sulfate.

[0040] (III) PCR method

[0041] The PCR reaction system was: 25 μL PrimeSTAR Max (purchased from Takara), 1 μL forward primer (10 μM), 1 μL reverse primer (10 μM), 1 μL template DNA, and 22 μL double distilled water. The PCR amplification conditions were: 98°C pre-denaturation for 5 min; followed by 30 cycles of 98°C for 10 s, 55°C for 15 s, and 72°C for 3 min, and finally 72°C for 5 min.

[0042] (IV) Method for preparing competent E. coli cells

[0043] E. coli competent cells were prepared using a Competent Cell Preparation Kit (purchased from Takara). The preparation method is described in the kit instructions.

[0044] (V) Plasmid chemical transformation of E. coli competent cells

[0045] The competent E. coli cells stored in the ultra-low temperature refrigerator were placed on ice to melt; 6 μL of PCR product was added and gently mixed, and ice bathed for 45 minutes. The centrifuge tube containing the competent cells was placed in a 42°C water bath for heat shock for 90 seconds, and the centrifuge tube was transferred to an ice bath to cool the cells for 2 minutes; 800 μL of LB culture medium was added and cultured at 37°C for 1 hour; a small amount of supernatant was removed by centrifugation, and the bacteria were resuspended, and the culture solution was spread on the LB plate containing the corresponding antibiotics, and inverted at 37°C for 10-12 hours. The colonies were observed, and a single colony was picked to verify the positive clone using colony PCR. The positive single colony was picked and cultured in LB liquid culture medium overnight, and the plasmid was extracted and sequenced for verification.

[0046] (VI) Preparation of electrocompetent cells of Corynebacterium glutamicum

[0047] (1) Corynebacterium glutamicum was inoculated into BHIS medium (selection was required on a freshly cultured slant, otherwise the growth of the bacteria would be affected), placed on a roving shaker (200 rpm), and cultured at 30°C for 16 h until the OD600 reached 3.0.

[0048] (2) Transfer the bacterial solution in (1) to the competent medium at an inoculum volume of 10% until the OD600 reaches 0.3, place it on a roving shaker (200 rpm), and culture it at 30°C until the OD600 reaches 0.9 (culture for about 3-5 hours, and it is in the logarithmic growth phase. Generally, if the bacterial concentration remains low at about 0.6, the subsequent operation can be continued). The general concentration multiple is 100 times (50 mL of competent medium is concentrated to 0.5 mL to prepare 5 tubes of competent cells).

[0049] (3) Place the bacterial solution in an ice-water bath for 15 min, centrifuge at 4000 rpm, 4°C for 10 min, and discard the supernatant.

[0050] (4) Fully suspend the cells in 30 mL of pre-cooled 10% glycerol, centrifuge at 4000 rpm, 4°C for 10 min, discard the supernatant, and repeat the washing and resuspending process four times.

[0051] (5) Resuspend the cells in 500 μL of pre-cooled 10% glycerol (concentrated 100 times) and dispense into 1.5 mL sterile centrifuge tubes, with 90-100 μL in each tube.

[0052] (6) Store the aliquoted competent cells at -80°C until use.

[0053] (VII) Electroporation transformation of Corynebacterium glutamicum

[0054] (1) Thaw the competent bacteria of Corynebacterium glutamicum stored at -80°C in an ice bath.

[0055] (2) Add 1-5.0 μL of plasmid, mix well, and place on ice for 5-10 min.

[0056] (3) Add to a pre-cooled 0.1 cm electric shock cup and shock twice at 1.8 kV for 5 ms.

[0057] (4) Quickly add 1.0 mL of culture medium (BHIS) preheated at 46°C, mix well, and transfer to a new 1.5 mL sterile centrifuge tube and incubate in a 46°C water bath for 6 min.

[0058] (5) Place the bacteria on a circulating shaker (220 rpm) and incubate at 30°C for 2 h.

[0059] (6) Centrifuge at 4000 rpm for 2 min at room temperature, discard part of the supernatant, resuspend the remaining bacterial solution and spread it on a solid plate coated with transformants with corresponding resistance, and culture in a constant temperature incubator at 30°C for 2-3 days.

[0060] (VIII) Recombinase efficiency test method

[0061] (1) Construction of plasmid pXMJ19 expressing endogenous recombinase

[0062] Genes CliR, CtiR, CauR, ChuR, CglR, recET, recA, and λred were synthesized using the Gene Synthesis Service of GENEWIZ. The linearized pXMJ19 plasmid was obtained by PCR amplification using the upstream primer (HP-pXMJ19-F) and the downstream primer (HP-pXMJ19-R). The target genes with homology arms were amplified by PCR using the primers CliR-F, CliR-R; CtiR-F, CtiR-R; CauR-F, CauR-R; ChuR-F, ChuR-R; CglR-F, CglR-R; recET-F, recET-R; recA-F, recA-R; λred-F, λred-R with the synthesized genes CliR, CtiR, CauR, ChuR, CglR, recET, recA, and λred templates, respectively. Then, the target genes with homology arms were amplified by PCR using the ClonExpress II One Step Cloning of Novozyme Biotech Co., Ltd. The expression vector and gene fragment were connected by using the Kit rapid cloning kit. The linearized vector obtained by PCR and the target gene fragment with homologous ends of the vector were recovered by gel and mixed at a molar ratio of 3 to 1. 5×CE II Buffer 4μL and Exnase II 2μL were added at the same time. Then ddH2O was added to make the total volume of the connection system reach 20μL. The reaction was carried out at 37℃ for 30min and then reduced to 4℃ for insulation. Then 10μL of the connection system was taken to transform Escherichia coli competent cells. The correct transformants of colony PCR were selected and sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing verification. Eight plasmids expressing different recombinases were obtained, namely pXMJ19-CliR, pXMJ19-CtiR, pXMJ19-CauR, pXMJ19-ChuR, pXMJ19-CglR, pXMJ19-recET, pXMJ19-recA, and pXMJ19-λred.

[0063] (2) Testing process of recombinase efficiency

[0064] We first constructed the initial strain to verify the efficiency of gene editing. We replaced the ldhA gene in the genome of Corynebacterium glutamicum S9114 with an inactivated Spc resistance gene with a nonsense sequence inserted in the middle; the Spc resistance gene with the amino acid at position 101 replaced with the TAA stop codon, respectively, called strains CgD and CgS. We used these two strains as a basis to verify the dsDNA recombination efficiency and ssDNA recombination efficiency of the recombinase. Specifically, we used strain CgD as a template, using the upstream primer dsDNA-FF and the downstream primer dsDNA-FR to obtain the upstream homologous arm of the nonsense fragment knocked out by PCR amplification, and the upstream primer dsDNA-RF and the downstream primer dsDNA-RR to obtain the downstream homologous arm of the nonsense fragment knocked out by PCR amplification. The upstream and downstream homologous arms were fused into dsDNA by the fusion PCR method. Then we mixed the constructed plasmid with homologous recombinase and dsDNA at a concentration of 500ng / ul: 500ng / ul, and aspirated 1000ng of the mixture into the strain CgD by electroporation. The number of colonies on the plate was used as a characterization method for editing efficiency. The knockout efficiency was then verified again by agarose gel electrophoresis. Similarly, the 90bp-long primer TAT-ssDNA synthesized by Jinweizhi was used as an ssDNA template to restore the activity of the inactivated Spc gene in the strain CgS. For specific test methods, see Figure 3 .

[0065]

[0066] 2. The sequence involved in the present invention is as follows:

[0067] <SEQ ID NO.1> Putative recombinase (CliR) from Corynebacterium liangguodongii:

[0068]

[0069] <SEQ ID NO.2> Putative recombinase from Corynebacterium timonense (CtiR):

[0070]

[0071] <SEQ ID NO.3> Putative recombinase (CauR) from Corynebacterium aurimucosum:

[0072]

[0073] <SEQ ID NO.4> Putative recombinase (ChuR) from Corynebacterium humireducens:

[0074]

[0075] Example 1 Screening method for iterative screening of endogenous recombinase based on Blast

[0076] The recET and λred systems have been proven to have high homologous recombination efficiency, but due to their poor compatibility in Corynebacterium glutamicum, we explored the endogenous recombinase of Corynebacterium based on the recET and λred recombinase systems. We used the amino acid sequences of recE, recT, and λredbeta as the initial sequences, respectively, and used the website https: / / www.ebi.ac.uk / Tools / hmmer / search / jackhmmer to perform blast searches in the Corynebacterium database. In the results of the first search, only the sequence based on recT searched for the endogenous recombinase, and the search based on other recombinases was unsuccessful. Afterwards, we used the results based on the first search as the initial sequence again, and iterated blast again (such as Figure 1 ). Among them, in the final results, we also identified a recE-like enzyme in front of the first four recT-like recombinases with higher scores in their genome. At the same time, we also identified a similar recT recombinase in the model bacterium Corynebacterium glutamicum ATCC 13032 (see the complete screening results for Figure 2 ).

[0077] Afterwards, we characterized the recombination efficiency of the five recombinases screened above (CliR, CtiR, CauR, ChuR, CglR) and the commonly used recET system, λred system, and recA. Figure 3 As shown. The recombinases from Corynebacterium liangguodongii (CliR), Corynebacterium timonense (CtiR), Corynebacterium aurimucosum (CauR), and Corynebacterium humireducens (ChuR) were tested for ssDNA-based homologous recombination efficiency and dsDNA-based homologous recombination efficiency, respectively; the recombinase from Corynebacterium glutamicum (CglR) was only tested for ssDNA homologous recombination efficiency because it did not recognize the recE structure.

[0078] The final results showed that the CauR endogenous recombinase had the greatest improvement in the gene editing efficiency of Corynebacterium glutamicum S9114, and no successful mutations were detected in ssDNA recombination based on the CglR endogenous recombinase. Finally, we also compared the sequences of the four recT-like enzymes we identified, such as Figure 4As shown, the screened recombinase has certain similarities with recT, which also proves its functional role.

[0079] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A gene editing system for Corynebacterium glutamicum, characterized in that: The gene editing system contains a recombinase, and the nucleotide sequence of the recombinase is shown in SEQ ID NO.

3.

2. The gene editing system according to claim 1, characterized in that: The Corynebacterium glutamicum includes Corynebacterium glutamicum S9114.

3. The gene editing system according to claim 1, characterized in that: The gene editing system also contains a dsDNA template and / or a ssDNA template.

4. A gene editing recombinase of Corynebacterium glutamicum, characterized in that The recombinase comprises the nucleotide sequence shown in SEQ ID NO.

3.

5. A vector system for gene editing of Corynebacterium glutamicum, characterized in that: The vector system contains an expression vector, and the expression vector is connected with a recombinase with a nucleotide sequence as shown in SEQ ID NO.

3.

6. The carrier system according to claim 5, characterized in that The expression vector includes the pXMJ19 plasmid.

7. A Corynebacterium glutamicum containing a recombinant enzyme, characterized in that The nucleotide sequence of the recombinase is shown in SEQ ID NO.

3.

8. A recombinase-mediated gene editing method for Corynebacterium glutamicum, characterized in that: The method comprises the step of introducing a recombinase into Corynebacterium glutamicum, wherein the nucleotide sequence of the recombinase is shown as SEQ ID NO.

3.

9. The gene editing method according to claim 8, characterized in that The recombinase is expressed episomally via a plasmid; Preferably, the plasmid contains a promoter for initiating the expression of the recombinase; Preferably, the promoter comprises an inducible promoter.

10. A method for screening gene editing recombinase for a target strain, characterized in that: The following steps are involved: S1. Using the known recombinase as the initial sequence, perform a blast search in the target strain database to obtain the blast result; S2. Use all the results of blast in S1 as the initial sequence, repeat step S1, and perform iterative search again until no recombinase with a higher score is screened out. Summarize all the identified recombinases, and the sequences with a score higher than 400 will be used for the gene editing recombinase of the target strain.

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