A recombinant Clostridium aerovorum and its construction method and application

Abstract

The invention provides a recombinant Clostridium aerovorum and a construction method and application thereof, belonging to the field of biotechnology. The Cas9 encoding gene is integrated into a choline utilization gene cluster of the Clostridium aerovorum, and the Clostridium aerovorum is Clostridium jundal or Clostridium autoethanogenum. The invention integrates the Cas9 encoding gene into the choline utilization gene cluster of the Clostridium aerovorum, and strictly regulates the expression of the Cas9 encoding gene through a choline-inducible promoter. The Cas9 transcription amount of the constructed recombinant Clostridium aerovorum during choline-added induction culture is significantly increased compared with that during culture without choline induction. Gene editing processes such as gene knock-in, knock-out and replacement can be achieved by introducing a plasmid containing only corresponding sgRNA and donor DNA, thereby shortening the construction time of the plasmid in the gene editing process, reducing the plasmid size of the CRISPR / Cas9 system, and improving the gene editing efficiency, which has an important role in promoting genetic research and metabolic engineering transformation of Clostridium aerovorum.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a recombinant Clostridium aerovorum and a construction method and application thereof. Background Art

[0002] Carbon-gas (such as CO2, CO) has huge reserves and is an important free carbon resource. It mainly comes from low-quality natural gas, tail gas emitted by petrochemical and smelting enterprises, and synthesis gas produced after biomass processing. Using carbon-fixing microorganisms for biological transformation is one of the main routes for carbon-gas resource utilization. It can effectively combine carbon fixation and emission reduction with green synthesis, which is of great significance for alleviating resource crises, reducing environmental pollution and promoting social sustainable development.

[0003] Clostridium aerovorum is a type of chemocarbon-fixing bacteria that can use syngas or industrial carbon-rich tail gas as the main carbon source. It is a Gram-positive anaerobic Clostridium. Clostridium aerovorum fixes CO / CO2 gas through the Wood-Ljungdahl pathway, which is converted into an important metabolic intermediate, acetyl-CoA, through a series of catalytic reactions. It then synthesizes various products such as acetic acid, ethanol, butanol, hexanol, etc. through various branch metabolic pathways. By metabolically engineering Clostridium aerovorum, more abundant non-natural products can also be produced. Therefore, Clostridium aerovorum is one of the ideal hosts for constructing a carbon-gas cell factory. For example, the invention patent with patent publication number "CN109072245A" (application number 201780021744.3) discloses a method for genetically engineering C1 fixed bacteria using CRISPR / Cas9 technology, where the C1 fixed bacteria are Clostridium autoethanogenum, Clostridium ljungdahlii or Clostridium ragsdalei.

[0004] The implementation of CRISPR / Cas9-based gene in-frame deletion operation technology in Clostridium aerogenes has been reported in the literature before. For example, the literature "Huang, et al., CRISPR / Cas9-Based Efficient Genome Editing in Clostridium ljungdahlii, an Autotrophic Gas-Fermenting Bacterium. ACS Synthetic Biology, 2016, 5, 1355-1361; Nagaraju et al., Genome editing of Clostridium autoethanogenum using CRISPR / Cas9. Biotechnol Biofuels, 2016 9: 219." discloses a CRISPR / Cas9-based gene in-frame deletion operation technology system. Compared with traditional tools, this technology system has the advantages of being faster, no need to add antibiotic resistance genes, and no scars. However, this technology system also has some problems, such as when introducing a gene encoding Cas9, sgRNA (single guide RNA) and donor at the same time, When a plasmid containing a DNA (gene editing template) is used, the plasmid length usually exceeds 10 kb, which will lead to low plasmid transformation efficiency and difficulty in achieving gene integration of larger fragments. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a recombinant Clostridium aerovorum and a construction method and application thereof, which can realize gene editing processes such as gene knockout, knockin, and replacement by introducing a plasmid containing only the corresponding sgRNA and donor DNA, thereby improving the gene editing efficiency.

[0006] The technical solution of the present invention is as follows:

[0007] A recombinant Clostridium aerovorum is obtained by integrating a Cas9 encoding gene into a choline utilization gene cluster of Clostridium aerovorum, wherein the nucleotide sequence of the Cas9 encoding gene is as described in SEQ ID NO.1, and the Clostridium aerovorum is Clostridium ljungdahlii or Clostridium autoethanogenum.

[0008] Preferably, the Clostridium jondahlii is Clostridium jondahlii DSM 13528.

[0009] Preferably, the Clostridium autoethanogenum is Clostridium autoethanogenum DSM 10061.

[0010] Further preferably, the introduction position of the Cas9 encoding gene in Clostridium ljungdahl DSM 13528 is between CP001666.1:4329750 and CP001666.1:4329751.

[0011] Further preferably, the introduction position of the Cas9 encoding gene in Clostridium autoethanogenum DSM 10061 is between CP006763.1:1964802 and CP006763.1:1964803.

[0012] The method for constructing the recombinant Clostridium aerovorum comprises the following steps:

[0013] S1, select a plasmid vector and construct a recombinant plasmid containing sgRNA and donor DNA, wherein the donor DNA contains a Cas9 encoding gene;

[0014] S2, introducing the recombinant plasmid in step S1 into Clostridium aerovorum;

[0015] S3, the recombinant plasmid is recombined with the Clostridium aerovorum genome through donor DNA, and the Cas9 encoding gene is integrated into the choline utilization gene cluster of the Clostridium aerovorum genome to obtain recombinant Clostridium aerovorum.

[0016] Wherein, the sgRNA targets the choline utilization gene cluster.

[0017] Among them, the Cas9 encoding gene is downstream of the choline-inducible promoter and is regulated by the choline-inducible promoter.

[0018] The application of the above recombinant plasmid is to use the recombinant plasmid to integrate the Cas9 encoding gene into the choline utilization gene cluster of the Clostridium aerovorum genome.

[0019] The use of the recombinant Clostridium aerovorum in gene editing of itself.

[0020] Preferably, the gene editing includes gene knock-in, gene knock-out, and gene replacement.

[0021] The genomes of Clostridium jundal and Clostridium autoethanogenum in Clostridium aerogenes both have a choline utilization gene cluster. Through experimental studies, we found that the choline utilization gene cluster contains a σ54 promoter, which is not only an inducible promoter, but also a stringent promoter. At the same time, the expression of the downstream genes of the choline utilization gene cluster will be upregulated by the induction of choline. If the Cas9 encoding gene is integrated into the choline utilization gene cluster of Clostridium jundal and Clostridium autoethanogenum, and the inducible promoter (σ54 promoter) is used to strictly regulate the expression of Cas9, gene editing can be performed by introducing a plasmid containing only the corresponding sgRNA and donor DNA, which greatly reduces the length of the plasmid, not only facilitating plasmid construction, but also making it possible to knock in or replace genes of larger fragments.

[0022] Beneficial effects:

[0023] (1) The present invention integrates the Cas9 encoding gene into the choline utilization gene cluster of Clostridium aerovorum, strictly regulates the expression of Cas9 through a choline-inducible promoter, and the Cas9 transcription amount of the constructed recombinant Clostridium aerovorum during choline-induced culture is significantly increased compared with that without choline induction. Gene editing processes such as gene knock-in, knock-out, and replacement can be achieved by introducing a plasmid containing only the corresponding sgRNA and donor DNA, shortening the plasmid construction time in the gene editing process, reducing the plasmid size of the CRISPR / Cas9 system, and improving the gene editing efficiency, which has an important role in promoting the genetic research and metabolic engineering transformation of Clostridium aerovorum.

[0024] (2) The present invention strictly regulates the expression of Cas9 through an inducible promoter, first constructs a recombinant Clostridium aerogenes containing a Cas9 encoding gene, and then performs gene editing on the basis of the recombinant Clostridium aerogenes, thereby greatly improving the transformation efficiency.

[0025] (3) When required for industrial applications, the Cas9 encoding gene can be deleted by introducing the corresponding sgRNA and donor DNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the result of characterizing the activity of the choline-inducible promoter using the CAT method.

[0027] Figure 2 This is the plasmid map of pMTLcas-KI.

[0028] Figure 3 This is a diagram showing the identification results of the recombinant strain of Clostridium Yongdal.

[0029] Figure 4 The Cas9 transcription level is different when cultured in PETC medium without choline and with choline.

[0030] Figure 5 This is a diagram to verify the knockout results of the riboflavin synthesis gene cluster.

[0031] Figure 6 This is a diagram for the verification of the CJ1586 gene knock-in results.

[0032] Figure 7 This is the verification diagram of the results of replacing the 2,3-butanediol dehydrogenase gene with the pdc gene.

[0033] Figure 8 This is the verification diagram of the CAETHG_1822-1823 gene knock-in results.

[0034] Fig. 9 Verification diagram of the results of restoring the recombinant strain of Clostridium Yongdal to the wild-type strain. DETAILED DESCRIPTION

[0035] The following is explained in conjunction with specific embodiments:

[0036] Source of experimental materials:

[0037] Clostridium jondahlii DSM 13528 and Clostridium autoethanogenum DSM 10061 were purchased from German Collection of Microorganisms and Cell Cultures.

[0038] Plasmid pMTL82254, plasmid pMTL82254: purchased from the University of Nottingham, UK; the above plasmids are recorded in the literature "Heap JT, Pennington OJ, Cartman ST, et al., A modular system for Clostridium shuttle plasmids. J Microbiol Methods. 2009.".

[0039] Plasmid pMTLcas-adhE1: recorded in the literature "Huang H, Chai C, Li N, et al. Crispr / cas9-based efficient genome editing in Clostridium ljungdahlii, an autotrophic gas-fermenting bacterium. ACS Synthetic Biology, 2016.", the above plasmid was donated by the research group.

[0040] Preparation of buffer and culture medium used:

[0041] SMP buffer: 270 mM sucrose, 1 mM magnesium chloride, 7 mM sodium phosphate, 10% DMSO, pH 6.0.

[0042] YTF liquid medium: yeast powder 10g / L, tryptone 16g / L, fructose 10g / L, sodium chloride 0.2g / L, L-cysteine ​​0.5g / L, 0.2% trace element storage solution (manganese sulfate monohydrate 5g / L, nickel chloride hexahydrate 0.2g / L, ammonium sulfate 5g / L, cobalt chloride hexahydrate 1g / L, zinc sulfate heptahydrate 1g / L, copper chloride dihydrate 0.2g / L), calcium chloride 0.2g / L, magnesium chloride 0.4g / L, sodium molybdate 0.0004g / L , sodium selenate 0.0004g / L, sodium tungstate 0.0004g / L, nitrilotriacetic acid 0.004g / L, resazurin 0.001g / L, ferrous sulfate 0.04g / L, 0.1% vitamin mixture (biotin 2mg / L, folic acid 2mg / L, vitamin B6 10mg / L, thiamine 5mg / L, riboflavin 5mg / L, calcium pantothenate 5mg / L, lipoic acid 5mg / L, p-aminobenzoic acid 5mg / L, cyanocobalamin 5mg / L, niacin 5mg / L).

[0043] YTF solid medium: Add 2% agar to YTF liquid medium.

[0044] PETC culture medium: macroelements (ammonium chloride 0.5 g / L, potassium chloride 0.2 g / L, magnesium sulfate heptahydrate 0.4 g / L, sodium chloride 1 g / L, potassium dihydrogen phosphate 0.2 g / L, sodium tungstate 0.005 g / L), trace metal elements (manganese chloride tetrahydrate 0.015 g / L, manganese sulfate monohydrate 0.015 g / L, ferrous sulfate 0.04 g / L, cobalt chloride hexahydrate 0.004 g / L, zinc sulfate heptahydrate 0.004 g / L) L, nickel chloride hexahydrate 0.0004g / L, sodium molybdate 0.004g / L, sodium selenate 0.002g / L), magnesium chloride 0.4g / L, calcium chloride monohydrate 0.02g / L, calcium chloride 0.2g / L, nitrilotriacetic acid 0.002g / L, resazurin 0.001g / L, yeast powder 1g / L, L-cysteine ​​0.25g / L, 0.1% vitamin mixture (biotin 2mg / L, folic acid 2mg / L, vitamin B6 10mg / L, thiamine 5mg / L, riboflavin 5mg / L, calcium pantothenate 5mg / L, lipoic acid 5mg / L, p-aminobenzoic acid 5mg / L, cyanocobalamin 5mg / L, niacin 5mg / L).

[0045] Example 1: Characterization of choline-inducible promoters

[0046] (1)pMTL82254-P CH Plasmid construction

[0047] ① Use restriction endonuclease NdeI to digest plasmid pMTL82254 and recover a linearized plasmid with a band size of about 6000 bp.

[0048] ② Using the genomic DNA of Clostridium jundal DSM 13528 as a template and CH-catP-F and CH-catP-R as primers, the choline-inducible promoter fragment P was amplified by PCR reaction. CH .

[0049] The nucleotide sequences of CH-catP-F and CH-catP-R are shown in SEQ ID NOs. 2 to 3, respectively.

[0050] ③Use 2× Fusein Mix to clone the linearized plasmid from step ① and the promoter fragment P from step ② of the recombinase assembly step CH , pMTL82254-P CH Plasmid.

[0051] (2)pMTL82254-P CH Construction of transformation strains

[0052] Frozen Clostridium jundal DSM 13528 was activated on ice, then transferred twice to YTF liquid medium and grown at 37°C until OD 600 Reach between 0.35 and 0.4. Add 0.1 times the volume of 2M sucrose solution and 0.15 times the volume of 1.25M glycine solution to the bacterial solution and incubate at 37°C for 2 hours. Centrifuge at 10000 rpm for 10 minutes at 4°C, collect the cell pellet, wash the cell pellet twice with SMP buffer and resuspend it to obtain competent cells for later use.

[0053] Electroporation (electroporation was performed in an anaerobic chamber): 200 μL of the competent cells were mixed with 4 μg of the pMTL82254-P CH The plasmids were mixed and incubated on ice for 1.5 min; the mixture was transferred to a 2 mm electroporation cup, and the electroporation parameters were set to 1.0 kV, 200 Ω, 50 μF to obtain the transformation solution.

[0054] The transformation solution was resuspended in 5 mL YTF liquid medium and incubated at 37°C for 14 h. 2.5 mL of the resuspended solution was centrifuged and the precipitate was spread on YTF solid medium containing 5 μg / mL clarithromycin and cultured at 37°C for about 3 days until a single colony grew out. The positive transformant, i.e., pMTL82254-P, was selected. CH Transformed strains.

[0055] (3)pMTL82254-P CH Analysis of catP expression level in transformed strains

[0056] pMTL82254-P CHThe transformed strain was inoculated into YTF liquid medium containing 5 μg / mL clarithromycin and cultured at 37°C for about 36 hours until the bacterial solution became turbid. The bacterial solution was inoculated into PETC medium containing 5 μg / mL clarithromycin and 5 g / L choline at a volume percentage of 10%, cultured at 37°C for 12 hours, and the cells were collected by centrifugation for detection of catP activity; at the same time, a control group was set up, that is, the bacterial solution was inoculated into PETC medium containing 5 μg / mL clarithromycin and 5 g / L fructose.

[0057] The catP activity was detected by DTNB method, which was described in the literature Nagaraju S, Davies NK, Walker DJ, M, Simpson SD. Genome editing of Clostridium autoethanogenum using CRISPR / Cas9. Biotechnol Biofuels. 2016》 was slightly modified. The specific method is as follows:

[0058] ① Prepare the mother solution required for the reaction detection solution: 0.1M TRIS-HCl buffer (pH 8.0), 2.5mg / mL chloramphenicol ethanol solution, 9.5mM acetyl-CoA aqueous solution, 8.33mM DTNB ethanol solution.

[0059] ②Prepare the reaction detection solution: mix TRIS-HCl buffer, chloramphenicol ethanol solution, acetyl-CoA aqueous solution, and DTNB ethanol solution in a volume ratio of 47:1:1:1 to obtain the reaction detection solution. The reaction detection solution should be prepared and used immediately.

[0060] ③ The collected bacteria were resuspended in TRIS-HCl buffer and ultrasonically disrupted to obtain a crude enzyme solution, and the protein concentration of the crude enzyme solution was detected by BCA method. 150 μL of reaction detection solution, 40 μL of TRIS-HCl buffer, and 10 μL of appropriately diluted crude enzyme solution were mixed, and the absorbance change (at 412 nm) within 60 seconds was detected on a microplate reader and the slope was calculated.

[0061] ④ Calculate the specific enzyme activity of the crude enzyme solution according to the following formula to measure catP activity:

[0062] In the formula, k A412 is the absorbance slope; df is the dilution factor of the crude enzyme solution; c is the protein concentration of the crude enzyme solution.

[0063] Tests and results Figure 1 As shown. Figure 1 Choline-induced pMTL82254-P CHThe catP activity of the strain increased 8.34 times compared with the control group, indicating that the choline-inducible promoter in Clostridium jondahlii DSM 13528 achieved the inducible expression of the catP gene.

[0064] Example 2: Construction of recombinant strains of Clostridium jundal

[0065] 1. Construction of Cas9 encoding gene (hereinafter referred to as “Cas9 gene”) integration plasmid

[0066] (1) Construction of pMTLcas-1 plasmid

[0067] ① Design guide RNA targeting the 20nt target region of the choline utilization gene cluster in Clostridium jundal DSM 13528 and design primers based on the guide RNA. The nucleotide sequence of the guide RNA is shown in SEQ ID NO.4.

[0068] ② Using pMTLcas-adhE1 plasmid as template and sgRNA-F and sgRNA-R as primers, sgRNA scaffold was amplified by PCR reaction.

[0069] Among them, the nucleotide sequences of primers sgRNA-F and sgRNA-R are shown in SEQ ID NOs.5 to 6, respectively.

[0070] The PCR reaction system was: 2×fastpfu Mix, 20 μL; sgRNA-F / sgRNA-R (10 μM), 1 μL each; pMTLcas-adhE1 plasmid 0.5 μL (50 ng); add water to make up to 40 μL.

[0071] PCR amplification conditions were as follows: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 15 sec, annealing at 55°C for 30 sec, extension at 72°C for 20 sec, 40 cycles; and extension at 72°C for 2 min.

[0072] ③Use restriction endonucleases SalI and XhoI to double digest plasmid pMTLcas-adhE1 and recover a band of about 9000 bp, which is the pMTLcas-adhE1 vector backbone.

[0073] ④ Use 2×Fusein Mix to clone the recombinase and assemble the sgRNA scaffold and pMTLcas-adhE1 vector backbone to obtain the pMTLcas-1 plasmid.

[0074] (2) Construction of pMTLcas-2 plasmid

[0075] ① Using the genomic DNA of Clostridium jungdal DSM 13528 as a template and LHA-F and LHA-R as primers, the homologous arm LHA located upstream of the insertion site was amplified by PCR reaction.

[0076] ② Using pMTLcas-adhE1 as a template and cas9-F1 and cas9-R1 as primers, the Cas9 gene fragment cas9-1 was amplified by PCR reaction.

[0077] Among them, the nucleotide sequences of primers LHA-F and LHA-R are shown in SEQ ID NOs.7 to 8, respectively, and the nucleotide sequences of primers cas9-F1 and cas9-R1 are shown in SEQ ID NOs.9 to 10, respectively.

[0078] The PCR reaction system was as follows: 2×fastpfu Mix, 20 μL; upstream and downstream primers (10 μM), 1 μL each; template 0.5 μL (50 ng); add water to make up to 40 μL;

[0079] PCR amplification conditions were as follows: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 15 sec, annealing at 55°C for 30 sec, extension at 72°C for 30 sec, 30 cycles; and extension at 72°C for 5 min.

[0080] ③ Assemble the homology arm LHA and the gene fragment cas9-1 through fusion PCR to obtain the upstream fragment.

[0081] The PCR reaction system was: 2×fastpfu Mix, 20 μL; 1 μL each of LHA-F / cas9-R1 primers (10 μM); 30 ng of LHA / cas9-1 fragment in total (molar ratio 1:1); and water was added to make up to 40 μL.

[0082] PCR amplification conditions were as follows: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 15 sec, annealing at 55°C for 30 sec, extension at 72°C for 30 sec, 30 cycles; and extension at 72°C for 5 min.

[0083] ④ The pMTLcas-1 plasmid was double digested with restriction endonucleases NotI and SacI to recover a band of about 9000 bp, which is the pMTLcas-1 vector backbone.

[0084] ⑤ Double digest the upstream fragment with restriction endonucleases NotI and SacI, and recover the digested fragment using the Cycle-Pure Kit.

[0085] ⑥ Use T4 ligase to connect the fragment digested in step ⑤ and the pMTLcas-1 vector backbone to obtain pMTLcas-2 plasmid.

[0086] (3) Construction of pMTLcas-KI plasmid

[0087] ① Using the genomic DNA of Clostridium jungdal DSM 13528 as a template and RHA-F and RHA-R as primers, the homologous arm RHA located downstream of the insertion site was amplified by PCR reaction.

[0088] ② Using pMTLcas-adhE1 as template and cas9-F2 and cas9-R2 as primers, the Cas9 gene fragment cas9-2 was amplified by PCR reaction.

[0089] Among them, the nucleotide sequences of primers RHA-F and RHA-R are shown in SEQ ID NOs.11 to 12, respectively, and the nucleotide sequences of primers cas9-F2 and cas9-R2 are shown in SEQ ID NOs.13 to 14, respectively.

[0090] The PCR reaction system was as follows: 2×fastpfu Mix, 20 μL; upstream and downstream primers (10 μM), 1 μL each; template 0.5 μL (50 ng); add water to make up to 40 μL;

[0091] PCR amplification conditions were as follows: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 15 sec, annealing at 55°C for 30 sec, extension at 72°C for 30 sec, 30 cycles; and extension at 72°C for 5 min.

[0092] ③ Assemble the homology arm RHA and the gene fragment cas9-2 through fusion PCR to obtain the downstream fragment.

[0093] The PCR reaction system was: 2×fastpfu Mix, 20 μL; 1 μL each of RHA-F / cas9-R2 primers (10 μM); 30 ng of RHA / cas9-2 fragment in total (molar ratio 1:1); and water was added to make up to 40 μL.

[0094] PCR amplification conditions were as follows: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 15 sec, annealing at 55°C for 30 sec, extension at 72°C for 30 sec, 30 cycles; and extension at 72°C for 5 min.

[0095] ④ The pMTLcas-2 plasmid was double digested with restriction endonucleases XbaI and NcoI to recover a band of about 9500 bp, which is the pMTLcas-2 vector backbone.

[0096] ⑤ Double digest the downstream fragment with restriction endonucleases XbaI and NcoI, and recover the digested fragment using the Cycle-Pure Kit.

[0097] ⑥Use T4 ligase to connect the fragment after enzyme digestion in step ⑤ and the pMTLcas-2 vector backbone to obtain pMTLcas-KI plasmid. The plasmid map is shown in Figure 2 shown.

[0098] 2. Construction of recombinant strains of Clostridium jundal

[0099] Frozen Clostridium jundal DSM 13528 was activated on ice, then transferred twice to YTF liquid medium and cultured at 37°C until the OD of the bacterial solution was 600 Between 0.35 and 0.4. Add 0.1 times the volume of 2M sucrose solution and 0.15 times the volume of 1.25M glycine solution to the bacterial solution and incubate at 37°C for 2 hours. Centrifuge at 10000 rpm for 10 minutes at 4°C, collect the cell pellet, wash the cell pellet twice with SMP buffer and resuspend it to obtain competent cells for later use.

[0100] Electroporation (the electroporation process was performed in an anaerobic chamber): 200 μL of the competent cells were mixed with 4 μg of the pMTLcas-KI plasmid and incubated on ice for 1.5 min; the mixture was transferred to a 2 mm electroporation cup, and the electroporation parameters were set to 1.0 kV, 200 Ω, 50 μF to obtain the transformation solution.

[0101] The transformation solution was resuspended in 5 mL of YTF liquid medium and incubated at 37°C for 14 h. 2.5 mL of the resuspended solution was centrifuged and the precipitate was spread on YTF solid medium containing 5 μg / mL thiamphenicol and cultured at 37°C for about 3 days until a single colony grew.

[0102] Pick a single colony and inoculate it into YTF liquid medium containing 5μg / mL thiamphenicol, and culture it at 37℃ for about 36h until the bacterial solution becomes turbid. Inoculate the bacterial solution into PETC medium containing 5g / L choline at a volume percentage of 10%, and culture it at 37℃ for 48h. Take the culture solution and streak it on YTF solid medium, and culture it at 37℃ for about 48h until a single colony grows.

[0103] Single colonies were picked and PCR was used to verify the insertion of the Cas9 gene. The primers used for verification were cas-test-F and cas-test-R. Positive clones were screened. The PCR band of positive clones was about 6400 bp, and that of the wild type was 2300 bp. The identification results were as follows: Figure 3 As shown, a band of about 6400 bp appeared in lane 2, and a positive clone was obtained, namely, a recombinant with Cas9 gene insertion.

[0104] The nucleotide sequences of primers cas-test-F and cas-test-R are shown in SEQ ID NOs. 15 to 16, respectively.

[0105] The Cas9 gene was inserted into the recombinant and continuously propagated in YTF solid culture medium without antibiotics to cause the pMTLcas-KI plasmid to be lost, thereby obtaining the plasmid-free target strain CLcas-KI, i.e., the recombinant strain of Clostridium jundal.

[0106] Example 3: Construction of recombinant strains of Clostridium autoethanogenum

[0107] 1. Construction of Cas9 gene integration plasmid (pMTLcas-KI-CA plasmid)

[0108] The downstream homology arm sequence and guide RNA sequence required in this example are completely consistent with the pMTLcas-KI plasmid, and only the LHA sequence needs to be replaced. The specific operation steps are as follows:

[0109] ① Using the genomic DNA of Clostridium autoethanogenum DSM 10061 as a template and LHA-F and LHA-R as primers, the homologous arm LHA located upstream of the insertion site was amplified by PCR reaction.

[0110] ② Using the pMTLcas-KI plasmid constructed in Example 2 as a template and cas9-F1 and cas9-R1 as primers, the Cas9 gene fragment cas9-1 was amplified by PCR reaction.

[0111] The PCR reaction system was as follows: 2×fastpfu Mix, 20 μL; upstream and downstream primers (10 μM), 1 μL each; template 0.5 μL (50 ng); add water to make up to 40 μL;

[0112] PCR amplification conditions were as follows: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 15 sec, annealing at 55°C for 30 sec, extension at 72°C for 30 sec, 30 cycles; and extension at 72°C for 5 min.

[0113] ③ Assemble the homology arm LHA and the gene fragment cas9-1 through fusion PCR to obtain the upstream fragment.

[0114] The PCR reaction system was as follows: 2×fastpfu Mix, 20 μL; 1 μL each of LHA-F / cas9-R1 primers (10 μM); 30 ng of LHA / cas9-1 fragment (molar ratio 1:1); add water to make up to 40 μL;

[0115] PCR amplification conditions were as follows: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 15 sec, annealing at 55°C for 30 sec, extension at 72°C for 30 sec, 30 cycles; and extension at 72°C for 5 min.

[0116] ④ The pMTLcas-KI plasmid was double digested with restriction endonucleases NotI and SacI to recover a band of about 11,000 bp, which is the pMTLcas-KI vector backbone.

[0117] ⑤ Double digest the upstream fragment with restriction endonucleases NotI and SacI, and recover the digested fragment using the Cycle-Pure Kit.

[0118] ⑥Use T4 ligase to connect the fragment digested in step ⑤ and the pMTLcas-KI vector backbone to obtain pMTLcas-KI-CA plasmid.

[0119] 2. Construction of recombinant strains of Clostridium autoethanogenum

[0120] The frozen Clostridium autoethanogenum DSM 10061 was activated on ice, then transferred twice to YTF liquid medium and cultured at 37°C until the OD of the bacterial solution reached 600 Between 0.35 and 0.4. Add 0.1 times the volume of 2M sucrose solution and 0.15 times the volume of 1.25M glycine solution to the bacterial solution and incubate at 37°C for 2 hours. Centrifuge at 10000 rpm for 10 minutes at 4°C, collect the cell pellet, wash the cell pellet twice with SMP buffer and resuspend it to obtain competent cells for later use.

[0121] Electroporation (the electroporation process was performed in an anaerobic chamber): 200 μL of the competent cells were mixed with 4 μg of the pMTLcas-KI-CA plasmid and incubated on ice for 1.5 min; the mixture was transferred to a 2 mm electroporation cup, and the electroporation parameters were set to 1.0 kV, 200 Ω, 50 μF to obtain the transformation solution.

[0122] The transformation solution was resuspended in 5 mL of YTF liquid medium and incubated at 37°C for 14 h. 2.5 mL of the resuspended solution was centrifuged and the precipitate was spread on YTF solid medium containing 5 μg / mL thiamphenicol and cultured at 37°C for about 3 days until a single colony grew.

[0123] Pick a single colony and inoculate it into YTF liquid medium containing 5μg / mL thiamphenicol, and culture it at 37℃ for about 36h until the bacterial solution becomes turbid. Inoculate the bacterial solution into PETC medium containing 5g / L choline at a volume percentage of 10%, and culture it at 37℃ for 48h. Take the culture solution and streak it on YTF solid medium, and culture it at 37℃ for about 48h until a single colony grows.

[0124] A single colony was picked and the Cas9 gene insertion result was verified by PCR. The primers used for verification were cas-test-F and cas-test-R (primer sequences were the same as above). Positive clones were screened. The PCR band of the positive clone was about 6400 bp, and that of the wild type was 2300 bp. The Cas9 gene-inserted recombinant was obtained.

[0125] The Cas9 gene was inserted into the recombinant and continuously propagated in YTF solid culture medium without antibiotics to cause the pMTLcas-KI plasmid to be lost, thereby obtaining the plasmid-free target strain CAcas-KI, that is, the recombinant strain of Clostridium autoethanogenum.

[0126] Example 4: Construction of gene knockout mutants using recombinant strains of Clostridium jundal

[0127] In this example, the riboflavin synthesis gene cluster (rib) in Clostridium jundal DSM 13528 is knocked out. The specific gene knockout steps are as follows:

[0128] 1. pMTL83151-P 1339 Plasmid construction

[0129] ① Design primers P1339-F and P1339-R to amplify P 1339 Promoter, used to regulate the expression of gRNA; wherein the nucleotide sequences of primers P1339-F and P1339-R are shown in SEQ ID NOs.17 to 18, respectively.

[0130] ② Using pMTLcas-KI plasmid as template and P1339-F and P1339-R as primers, P was amplified by PCR reaction. 1339 Promoter.

[0131] ③ Insert pMTL83151 plasmid and P 1339 The promoter fragment was double digested with restriction endonucleases SalI and XbaI, and a band of about 4500 bp was recovered from the pMTL83151 plasmid, which was the pMTL83151 vector backbone; 1339 The promoter fragment was recovered using the Cycle-Pure Kit.

[0132] ④ Use T4 ligase to connect the pMTL83151 vector backbone and promoter fragment P 1339 , pMTL83151-P 1339 Plasmid.

[0133] 2. Construction of pMTL83151-Δrib plasmid

[0134] ① Design guide RNA targeting the 20nt target region of the riboflavin biosynthesis gene cluster (CP001666.1:2407831-2411585) in Clostridium jundal DSM 13528. The guide RNA sequence is shown in SEQ ID NO.19. Primers rib-sgRNA-F and sgRNA-R2 are designed based on the guide RNA.

[0135] Among them, the nucleotide sequences of primers rib-sgRNA-F and sgRNA-R2 are shown in SEQ ID NOs. 20 to 21, respectively.

[0136] ② Using pMTLcas-adhE1 as a template and rib-sgRNA-F and sgRNA-R2 as primers, the rib-sgRNA scaffold was amplified by PCR reaction.

[0137] ③ Design primers rib-LHA-F, rib-LHA-R and rib-RHA-F, rib-RHA-R to amplify the homology arms on both sides of the riboflavin biosynthesis gene cluster. The nucleotide sequences of the primers are shown in SEQ ID NOs. 22 to 25, respectively.

[0138] ④ Using the genomic DNA of Clostridium Jondal DSM 13528 as a template and rib-LHA-F and rib-LHA-R as primers, the upstream homology arm rib-LHA was amplified by PCR reaction; using the genomic DNA of Clostridium Jondal DSM 13528 as a template and rib-RHA-F and rib-RHA-R as primers, the downstream homology arm rib-RHA was amplified by PCR reaction.

[0139] ⑤ Assemble the rib-sgRNA scaffold and the upstream homology arm rib-LHA through fusion PCR to obtain the gene fragment rib-sgRNA-LHA.

[0140] ⑥PMTL83151-P 1339 The plasmid was double digested with restriction endonucleases SalI and XhoI, and a band of about 4700 bp was recovered, namely pMTL83151-P 1339 Carrier skeleton.

[0141] ⑦Use 2×Fusein Mix to clone the recombinase assembly gene fragment rib-sgRNA-LHA, downstream homology arm rib-RHA, pMTL83151-P 1339 The vector backbone was used to obtain the pMTL83151-Δrib plasmid.

[0142] 3. Construction of riboflavin biosynthesis gene cluster knockout mutants

[0143] The frozen recombinant strain of Clostridium jundal CLcas-KI was activated on ice, then transferred twice in YTF liquid medium and grown at 37°C until OD 600 Between 0.35 and 0.4. Add 0.1 times the volume of 2M sucrose solution and 0.15 times the volume of 1.25M glycine solution to the bacterial solution and incubate at 37°C for 2 hours. Centrifuge at 10000 rpm for 10 minutes at 4°C, collect the cell pellet, wash the cell pellet twice with SMP buffer and resuspend it to obtain competent cells for later use.

[0144] Electroporation (the electroporation process was performed in an anaerobic chamber): 200 μL of the competent cells were mixed with 4 μg of the pMTL83151-Δrib plasmid and incubated on ice for 1.5 min; the mixture was transferred to a 2 mm electroporation cup, and the electroporation parameters were set to 1.0 kV, 200 Ω, 50 μF to obtain the transformation solution.

[0145] The transformation solution was resuspended in 5 mL of YTF liquid medium and incubated at 37°C for 14 h. 2.5 mL of the resuspended solution was centrifuged and the precipitate was spread on YTF solid medium containing 5 μg / mL thiamphenicol and cultured at 37°C for about 3 days until a single colony grew.

[0146] Pick a single colony and inoculate it into YTF liquid medium containing 5 μg / mL thiamphenicol, and culture it at 37°C for about 36 hours until the bacterial solution becomes turbid. Inoculate the bacterial solution into PETC medium containing 5 g / L choline and 5 μg / mL thiamphenicol at a volume percentage of 10% (PETC medium without choline was used as a control), and induce culture at 37°C for 48 hours.

[0147] The difference in Cas9 transcription levels between cells cultured in PETC medium without choline and with choline is shown in Figure 4 As shown. Figure 4 It can be found that the transcription level of Cas9 gene in the recombinant strain CLcas-KI of Clostridium Jungdalar constructed in Example 2 was increased by about 12 times when choline was added for induction culture compared with that without choline induction. This indicates that the introduction of Cas9 gene into the choline utilization gene cluster of Clostridium Jungdalar can strictly regulate the expression of Cas9 gene through the choline-inducible promoter.

[0148] After 48 hours of induction culture at 37℃, the culture solution was streaked on YTF solid medium and cultured at 37℃ for about 48 hours until a single colony grew out. A single colony was picked and the gene knockout result was verified by PCR. The primers used for identification were rib-test-F and rib-test-R. Positive clones were screened. The PCR band of positive clones was about 2000bp, and that of wild type was 5800bp. The identification results were as follows: Figure 5 As shown, a band of about 2000 bp appeared in lane 2, which was a positive clone.

[0149] The nucleotide sequences of primers rib-test-F and rib-test-R are shown in SEQ ID NOs. 26 to 27, respectively.

[0150] The PCR products of the positive clones were further sequenced for verification, and the sequencing results showed that the riboflavin synthesis gene cluster was missing, indicating that the gene knockout mutant strain Δrib was successfully obtained.

[0151] Example 5: Construction of gene knock-in mutants using recombinant strains of Clostridium jundal

[0152] The hemoglobin gene CJ1586 from Campylobacter jejuni was codon optimized and commercially synthesized to obtain the pUC-CJ1586 plasmid; the codon-optimized CJ1586 gene was knocked into the genome of Clostridium jundal DSM 13528 at CP001666.1:1010558-1010559 to form a polycistronic with CLJU_c09100 and CLJU_c09110. The specific gene knock-in steps are as follows:

[0153] 1. Construction of pMTL83151-CJ1586-KI plasmid

[0154] ① Design guide RNA targeting the 20nt target region CLJU_c09100 of the genome of Clostridium jundal DSM 13528, the guide RNA sequence is shown in SEQ ID NO.28, and design primer CJ1586-sgRNA-F based on the guide RNA, and the primer nucleotide sequence is shown in SEQ ID NO.29.

[0155] ② Using pMTLcas-adhE1 as a template and CJ1586-sgRNA-F and sgRNA-R2 as primers, the CJ1586-sgRNA scaffold was amplified by PCR reaction.

[0156] ③ Design primers CJ1586-LHA-F1, CJ1586-LHA-R1 and CJ1586-LHA-F2, CJ1586-LHA-R2 and CJ1586-RHA-F, CJ1586-RHA-R to amplify the homology arms on both sides of the insertion site. The sequences on the homology arms corresponding to the guide RNA are designed as synonymous mutations; design primers CJ1586-F and CJ1586-R to amplify the codon-optimized CJ1586 gene.

[0157] The designed primer sequences are shown in SEQ ID NOs. 30 to 37 respectively.

[0158] ④ Using the genomic DNA of Clostridium jundal DSM 13528 as a template and CJ1586-LHA-F1 and CJ1586-LHA-R1 as primers, the first segment of the upstream homology arm CJ1586-LHA1 was amplified by PCR reaction; using the genomic DNA of Clostridium jundal DSM 13528 as a template and CJ1586-LHA-F2 and CJ1586-LHA-R2 as primers, the second segment of the upstream homology arm CJ1586-LHA2 was amplified by PCR reaction; using the genomic DNA of Clostridium jundal DSM 13528 as a template and CJ1586-LHA-F2 and CJ1586-LHA-R2 as primers, the second segment of the upstream homology arm CJ1586-LHA2 was amplified by PCR reaction; Using 13528 genomic DNA as a template and CJ1586-RHA-F and CJ1586-RHA-R as primers, the downstream homology arm CJ1586-RHA was amplified by PCR reaction; using pUC-CJ1586 plasmid as a template and CJ1586-F and CJ1586-R as primers, the gene fragment CJ1586 was amplified by PCR reaction.

[0159] ⑤ Assemble the CJ1586-sgRNA scaffold and the first segment of the upstream homology arm CJ1586-LHA1 through fusion PCR to obtain the gene fragment CJ1586-sgRNA-LHA; assemble the second segment of the upstream homology arm CJ1586-LHA2 and the gene fragment CJ1586 through fusion PCR to obtain the gene fragment LHA-CJ1586.

[0160] ⑥PMTL83151-P 1339 The plasmid was double digested with restriction endonucleases SalI and XhoI, and a band of about 4700 bp was recovered, namely pMTL83151-P 1339 Carrier skeleton.

[0161] ⑦Use 2×Fusein Mix to clone recombinase to assemble gene fragment CJ1586-sgRNA-LHA, gene fragment LHA-CJ1586, downstream homology arm CJ1586-RHA, pMTL83151-P 1339 The vector backbone was removed to obtain the pMTL83151-CJ1586-KI plasmid.

[0162] 2. Construction of CJ1586 knock-in mutant

[0163] The frozen recombinant strain of Clostridium jundal CLcas-KI was activated on ice, then transferred twice in YTF liquid medium and grown at 37°C until OD 600 Between 0.35 and 0.4. Add 0.1 times the volume of 2M sucrose solution and 0.15 times the volume of 1.25M glycine solution to the bacterial solution and incubate at 37°C for 2 hours. Centrifuge at 10000 rpm for 10 minutes at 4°C, collect the cell pellet, wash the cell pellet twice with SMP buffer and resuspend it to obtain competent cells for later use.

[0164] Electroporation (the electroporation process was performed in an anaerobic chamber): 200 μL of the competent cells were mixed with 4 μg of the pMTL83151-CJ1586-KI plasmid and incubated on ice for 1.5 min; the mixture was transferred to a 2 mm electroporation cup, and the electroporation parameters were set to 1.0 kV, 200 Ω, 50 μF to obtain the transformation solution.

[0165] The transformation solution was resuspended in 5 mL of YTF liquid medium and incubated at 37°C for 14 h. 2.5 mL of the resuspended solution was centrifuged and the precipitate was spread on YTF solid medium containing 5 μg / mL thiamphenicol and cultured at 37°C for about 3 days until a single colony grew.

[0166] Pick a single colony and inoculate it into YTF liquid medium containing 5 μg / mL thiamphenicol, and culture it at 37°C for about 36 hours until the bacterial solution becomes turbid. Inoculate the bacterial solution into PETC medium containing 5 g / L choline and 5 μg / mL thiamphenicol at a volume percentage of 10%, and induce the culture at 37°C for 48 hours.

[0167] Take the culture medium and streak it on YTF solid medium, and culture it at 37℃ for about 48h until a single colony grows. Pick a single colony and use PCR to verify the gene knock-in result. The primers used for identification are CJ1586-test-F and CJ1586-test-R. Screen positive clones. The PCR band of positive clones is about 2400bp, and that of wild type is 2000bp. The identification results are as follows: Figure 6 As shown, a band of about 2400 bp appeared in lane 2, which was a positive clone.

[0168] The nucleotide sequences of primers CJ1586-test-F and CJ1586-test-R are shown in SEQ ID NOs. 38 to 39, respectively.

[0169] The PCR products of the positive clones were further sequenced and verified, and the sequencing results showed that the CJ1586 gene was successfully knocked in, that is, the gene knock-in mutant strain CJ1586-KI was successfully obtained.

[0170] Example 6: Construction of gene replacement mutants using recombinant strains of Clostridium jundal

[0171] The pyruvate decarboxylase gene pdc from Zymomonas mobilis subsp. mobilis ATCC 29191 was codon optimized and commercially synthesized to obtain the pUC-pdc plasmid; the codon-optimized pdc gene was used to replace the 2,3-butanediol dehydrogenase gene (CLJU_c23220) in the genome of Clostridium jundal DSM 13528. The specific gene replacement steps are as follows:

[0172] 1. Construction of pMTL83151-Δbdh::pdc plasmid

[0173] ① Design guide RNA targeting the 20nt target region of the bdh gene (CLJU_c23220) in the genome of Clostridium jundal DSM 13528, the guide RNA sequence is shown in SEQ ID NO.40, and design primer pdc-sgRNA-F based on the guide RNA, and the primer nucleotide sequence is shown in SEQ ID NOs.41.

[0174] ② Using pMTLcas-adhE1 as a template and pdc-sgRNA-F and sgRNA-R2 as primers, the pdc-sgRNA scaffold was amplified by PCR reaction.

[0175] ③ Design primers pdc-LHA-F, pdc-LHA-R and pdc-RHA-F, pdc-RHA-R to amplify the homology arms on both sides of CLJU_c23220; design primers pdc-F and pdc-R to amplify the codon-optimized pdc gene.

[0176] The designed primer sequences are shown in SEQ ID NOs. 42 to 47 respectively.

[0177] ④ Using the genomic DNA of Clostridium Jondal DSM 13528 as a template, pdc-LHA-F and pdc-LHA-R as primers, the upstream homology arm pdc-LHA was amplified by PCR reaction; using the genomic DNA of Clostridium Jondal DSM 13528 as a template, pdc-RHA-F and pdc-RHA-R as primers, the downstream homology arm pdc-RHA was amplified by PCR reaction; using the pUC-pdc plasmid as a template, pdc-F and pdc-R as primers, the gene fragment pdc was amplified by PCR reaction.

[0178] ⑤ Assemble the pdc-sgRNA scaffold and the upstream homology arm pdc-LHA by fusion PCR to obtain the gene fragment pdc-sgRNA-LHA;

[0179] ⑥PMTL83151-P 1339 The plasmid was double digested with restriction endonucleases SalI and XhoI, and a band of about 4700 bp was recovered, namely pMTL83151-P 1339 Carrier skeleton.

[0180] ⑦Use 2×Fusein Mix to clone recombinase to assemble gene fragment pdc-sgRNA-LHA, downstream homology arm pdc-RHA, gene fragment pdc, pMTL83151-P 1339 Vector backbone to obtain pMTL83151-Δbdh::pdc plasmid.

[0181] 2. Construction of Δbdh::pdc gene replacement mutant

[0182] The frozen recombinant strain of Clostridium jundal CLcas-KI was activated on ice, then transferred twice in YTF liquid medium and grown at 37°C until OD 600 Between 0.35 and 0.4. Add 0.1 times the volume of 2M sucrose solution and 0.15 times the volume of 1.25M glycine solution to the bacterial solution and incubate at 37°C for 2 hours. Centrifuge at 10000 rpm for 10 minutes at 4°C, collect the cell pellet, wash the cell pellet twice with SMP buffer and resuspend it to obtain competent cells for later use.

[0183] Electroporation (the electroporation process was performed in an anaerobic chamber): 200 μL of the competent cells were mixed with 4 μg of the plasmid pMTL83151-Δbdh::pdc on ice and incubated for 1.5 min; the mixture was transferred to a 2 mm electroporation cup, and the electroporation parameters were set to 1.0 kV, 200 Ω, 50 μF to obtain the transformation solution.

[0184] The transformation solution was resuspended in 5 mL of YTF liquid medium and incubated at 37°C for 14 h. 2.5 mL of the resuspended solution was centrifuged and the precipitate was spread on YTF solid medium containing 5 μg / mL thiamphenicol and cultured at 37°C for about 3 days until a single colony grew.

[0185] Pick a single colony and inoculate it into YTF liquid medium containing 5 μg / mL thiamphenicol, and culture it at 37°C for about 36 hours until the bacterial solution becomes turbid. Inoculate the bacterial solution into PETC medium containing 5 g / L choline and 5 μg / mL thiamphenicol at a volume percentage of 10%, and induce the culture at 37°C for 48 hours.

[0186] Take the culture medium and streak it on YTF solid medium, and culture it at 37℃ for about 48 hours until a single colony grows. Pick a single colony and use PCR to verify the gene replacement result. The primers used for identification are pdc-test-F and pdc-test-R. Screen positive clones. The PCR band of positive clones is about 3200bp, and that of wild type is 2600bp. The identification results are as follows: Figure 7 As shown, a band of about 3200 bp appeared in lane 2, which was a positive clone.

[0187] The nucleotide sequences of primers pdc-test-F and pdc-test-R are shown in SEQ ID NOs. 48 to 49, respectively.

[0188] The PCR products of the positive clones were further sequenced for verification, and the sequencing results showed that the 2,3-butanediol dehydrogenase gene had been replaced by the pdc gene, that is, the gene replacement mutant strain Δbdh::pdc was successfully obtained.

[0189] Example 7: Construction of gene knock-in mutants using recombinant strains of Clostridium autoethanogenum

[0190] The CAETHG_1822-1823 gene of Clostridium autoethanogenum DSM 10061 was knocked into the genome of Clostridium autoethanogenum DSM 10061 at CP006763.1:3539285-3935286. The specific gene knock-in steps are as follows:

[0191] 1. Construction of pMTL83151-1823-KI plasmid

[0192] ① Design guide RNA targeting the insertion site of Clostridium autoethanogenum DSM 10061, the guide RNA sequence is shown in SEQ ID NO.50, and primer 1823-sgRNA-F is designed based on the guide RNA, and the primer nucleotide sequence is shown in SEQ ID NO.51.

[0193] ② Using pMTLcas-adhE1 as a template and 1823-sgRNA-F and sgRNA-R2 as primers, the 1823-sgRNA scaffold was amplified by PCR reaction.

[0194] ③ Primers 1823-LHA-F, 823-LHA-R and 1823-RHA-F, 1823-RHA-R were designed to amplify the homology arms on both sides of the insertion site. The sequences on the homology arms corresponding to the guide RNA were designed as synonymous mutations; primers 1823-F and 1823-R were designed to amplify the CAETHG_1822-1823 gene.

[0195] The designed primer sequences are shown in SEQ ID NOs. 52 to 57 respectively.

[0196] ④ Using the genomic DNA of Clostridium autoethanogenum DSM 10061 as a template and 1823-LHA-F and 1823-LHA-R as primers, the upstream homologous arm 1823-LHA was amplified by PCR reaction; using the genomic DNA of Clostridium autoethanogenum DSM 10061 as a template and 1823-RHA-F and 1823-RHA-R as primers, the downstream homologous arm 1823-RHA was amplified by PCR reaction; using the genomic DNA of Clostridium autoethanogenum DSM 10061 as a template and 1823-F and 1823-R as primers, the gene fragment 1823 was amplified by PCR reaction.

[0197] ⑤ Assemble the 1823-sgRNA scaffold and the upstream homology arm 1823-LHA through fusion PCR to obtain the gene fragment 1823-sgRNA-LHA.

[0198] ⑥PMTL83151-P 1339 The plasmid was double digested with restriction endonucleases SalI and XhoI, and a band of about 4700 bp was recovered, namely pMTL83151-P 1339 Carrier skeleton.

[0199] ⑦Use 2×Fusein Mix to clone recombinase to assemble gene fragment 1823-sgRNA-LHA, gene fragment 1823, downstream homology arm 1823-RHA, pMTL83151-P 1339 The vector backbone was used to obtain the pMTL83151-1823-KI plasmid.

[0200] 2. Construction of CAETHG_1822-1823 gene knock-in mutant

[0201] The frozen recombinant strain of Clostridium autoethanogenum CAcas-KI was activated on ice, then transferred twice to YTF liquid medium and grown at 37°C until OD 600 Between 0.35 and 0.4. Add 0.1 times the volume of 2M sucrose solution and 0.15 times the volume of 1.25M glycine solution to the bacterial solution and incubate at 37°C for 2 hours. Centrifuge at 10000 rpm for 10 minutes at 4°C, collect the cell pellet, wash the cell pellet twice with SMP buffer and resuspend it to obtain competent cells for later use.

[0202] Electroporation (the electroporation process was performed in an anaerobic chamber): 200 μL of the competent cells were mixed with 4 μg of the pMTL83151-1823-KI plasmid on ice and incubated for 1.5 min; the mixture was transferred to a 2 mm electroporation cup, and the electroporation parameters were set to 1.0 kV, 200 Ω, 50 μF to obtain the transformation solution.

[0203] The transformation solution was resuspended in 5 mL of YTF liquid medium and incubated at 37°C for 14 h. 2.5 mL of the resuspended solution was centrifuged and the precipitate was spread on YTF solid medium containing 5 μg / mL thiamphenicol and cultured at 37°C for about 3 days until a single colony grew.

[0204] Pick a single colony and inoculate it into YTF liquid medium containing 5 μg / mL thiamphenicol, and culture it at 37°C for about 36 hours until the bacterial solution becomes turbid. Inoculate the bacterial solution into PETC medium containing 5 g / L choline and 5 μg / mL thiamphenicol at a volume percentage of 10%, and induce the culture at 37°C for 48 hours.

[0205] Take the culture medium and streak it on YTF solid medium, and culture it at 37℃ for about 48h until a single colony grows. Pick a single colony and use PCR to verify the gene knock-in result. The primers used for identification are 1823-test-F and 1823-test-R. Screen positive clones. The PCR band of positive clones is about 4600bp, and that of wild type is 2700bp. The identification results are as follows: Figure 8 As shown, a band of about 4600 bp appeared in lane 2, which was a positive clone.

[0206] The nucleotide sequences of primers 1823-test-F and 1823-test-R are shown in SEQ ID NOs. 58 to 59, respectively.

[0207] The PCR products of the positive clones were further sequenced for verification, and the sequencing results showed that the CAETHG_1822-1823 gene was successfully knocked in, that is, the gene knock-in mutant strain 1822-1823-KI was successfully obtained.

[0208] Example 8: Construction of recombinant strains of Clostridium jundal restored to wild-type strains

[0209] In this example, the Cas9 gene introduced into the recombinant strain of Clostridium jundal is removed to restore the wild-type strain. The specific steps are as follows:

[0210] 1. Construction of pMTL83151-cas-KO plasmid

[0211] ① Design guide RNA targeting Cas9 gene, the guide RNA sequence is shown in SEQ ID NO.60, and design primer cas-sgRNA-F based on the guideRNA, and the primer nucleotide sequence is shown in SEQ ID NO.61.

[0212] ② Using pMTLcas-adhE1 as a template and cas-sgRNA-F and sgRNA-R2 as primers, the cas-sgRNA scaffold was amplified by PCR reaction.

[0213] ③ Design primers cas-HA-F and cas-HA-R for amplifying homology arms, and the nucleotide sequences of the primers are shown in SEQ ID NOs.62 to 63, respectively.

[0214] ④ Using the genomic DNA of Clostridium jungdal DSM 13528 as a template and cas-HA-F and cas-HA-R as primers, the homologous arm cas-HA was amplified by PCR reaction.

[0215] ⑤ Insert pMTL83151-P 1339 The plasmid was double digested with restriction endonucleases SalI and XhoI, and a band of about 4700 bp was recovered, namely pMTL83151-P 1339 Carrier skeleton.

[0216] ⑥Use 2×Fusein Mix to clone recombinase to assemble cas-sgRNA scaffold, homology arm cas-HA fragment, pMTL83151-P 1339 Vector backbone to obtain pMTL83151-cas-KO plasmid.

[0217] 2. Construction of wild-type strain

[0218] The frozen recombinant strain of Clostridium jundal CLcas-KI was activated on ice, then transferred twice in YTF liquid medium and grown at 37°C until OD 600 Between 0.35 and 0.4. Add 0.1 times the volume of 2M sucrose solution and 0.15 times the volume of 1.25M glycine solution to the bacterial solution and incubate at 37°C for 2 hours. Centrifuge at 10000 rpm for 10 minutes at 4°C, collect the cell pellet, wash the cell pellet twice with SMP buffer and resuspend it to obtain competent cells for later use.

[0219] Electroporation (the electroporation process was performed in an anaerobic chamber): 200 μL of the competent cells were mixed with 4 μg of the pMTL83151-cas-KO plasmid and incubated on ice for 1.5 min; the mixture was transferred to a 2 mm electroporation cup, and the electroporation parameters were set to 1.0 kV, 200 Ω, 50 μF to obtain the transformation solution.

[0220] The transformation solution was resuspended in 5 mL of YTF liquid medium and incubated at 37°C for 14 h. 2.5 mL of the resuspended solution was centrifuged and the precipitate was spread on YTF solid medium containing 5 μg / mL thiamphenicol and cultured at 37°C for about 3 days until a single colony grew.

[0221] Pick a single colony and inoculate it into YTF liquid medium containing 5 μg / mL thiamphenicol, and culture it at 37°C for about 36 hours until the bacterial solution becomes turbid. Inoculate the bacterial solution into PETC medium containing 5 g / L choline and 5 μg / mL thiamphenicol at a volume percentage of 10%, and induce the culture at 37°C for 48 hours.

[0222] Take the culture medium and streak it on YTF solid medium, and culture it at 37℃ for about 48h until a single colony grows. Pick a single colony and use PCR to verify the result of Cas9 gene knockout. The primers used for identification are cas-test-F2 and cas-test-R2, and the primer sequences are shown in SEQ ID NOs.64-65 respectively. Screen the positive clones. The starting strain band is about 6200bp, and the PCR band of the positive clone is about 2000bp. The identification result is as follows Fig. 9 As shown, a band of about 2000 bp appeared in lane 2, which was a positive clone.

[0223] The PCR products of the positive clones were further sequenced and verified, and the sequencing results showed that the Cas9 gene was missing, indicating that the wild-type strain was successfully restored.

Claims

1. A recombinant Clostridium aerovorum, characterized in that: The Cas9 encoding gene is integrated into the choline utilization gene cluster of Clostridium aerovorum, wherein the nucleotide sequence of the Cas9 encoding gene is as shown in SEQ ID NO.1, and the Clostridium aerovorum is Clostridium jundal ( Clostridium ljungdahlii ) or Clostridium autoethanogenum ( Clostridium autoethanogenum ); the Clostridium jondahlii is Clostridium jondahlii DSM 13528, and the Clostridium autoethanogenum is Clostridium autoethanogenum DSM 10061; Wherein, the choline utilization gene cluster contains a choline-inducible promoter, and the Cas9 encoding gene is downstream of the choline-inducible promoter and is regulated by the choline-inducible promoter; The introduction position of the Cas9 encoding gene in Clostridium jondahlii DSM 13528 is between CP001666.1: 4329750 and CP001666.1: 4329751, and the introduction position of the Cas9 encoding gene in Clostridium autoethanogenum DSM 10061 is between CP006763.1: 1964802 and CP006763.1: 1964803.

2. The method for constructing the recombinant Clostridium aerovorum according to claim 1, characterized in that: The steps include: S1, select a plasmid vector and construct a recombinant plasmid containing sgRNA and donor DNA, wherein the donor DNA contains a Cas9 encoding gene; S2, introducing the recombinant plasmid in step S1 into Clostridium aerovorum; S3, the recombinant plasmid is recombined with the Clostridium aerovorum genome through donor DNA, and the Cas9 encoding gene is integrated into the choline utilization gene cluster of the Clostridium aerovorum genome to obtain recombinant Clostridium aerovorum.

3. The construction method according to claim 2, characterized in that: The sgRNA targets the choline utilization gene cluster.

4. The use of the recombinant Clostridium aerovorum according to claim 1, characterized in that: Used for self-gene editing.

5. The use according to claim 4, characterized in that The gene editing includes gene knock-in, gene knock-out, and gene replacement.

6. An application of a recombinant plasmid, characterized in that: The recombinant plasmid is used to integrate the Cas9 encoding gene into the choline utilization gene cluster of the Clostridium aerovorum genome; wherein the recombinant plasmid is prepared according to the construction method described in claim 2.

Images