Ketogulonigenium vulgare gene inhibition system and application thereof

Abstract

The invention relates to a ketogulonigenium vulgare gene inhibition system and application thereof, and belongs to the technical field of gene engineering. The invention solves the problems of low gene editing efficiency and poor applicability of the ketogulonigenium sp., adopts the CRISPR Cas12 related dCpf1 protein to construct a gene inhibition system, provides a new method for inhibiting the target gene in the ketogulonigenium sp., can quickly and effectively inhibit the expression of the target gene, and has a good application prospect in the field of ketogulonigenium sp. And the gene editing efficiency of the ketogulonigenium sp. Is improved. The ketogulonigenium vulgare endogenous promoter P1066 is screened through multiple experiments, the promoter can be stably and efficiently transcribed in a strain, dCpf1 coding gene expression can be accurately driven, the problems that an exogenous promoter is poor in adaptability and high in off-target rate are solved, and the specificity and reliability of gene inhibition are guaranteed.

Description

Technical Field

[0001] This invention relates to a ketogenic gulonic acid bacteria gene inhibition system and its application, belonging to the field of genetic engineering technology. Background Technology

[0002] Keto-producing gulonic acid bacteria are one of the classic two-step fermentation strains for vitamin C production, and also the most promising strains for one-step vitamin C fermentation research. In the study of the metabolic network related to 2-keto-L-gulonic acid (2-KGA, a vitamin C precursor) in keto-producing gulonic acid bacteria, the development of gene editing techniques has significant application value. For example, Chinese patent document CN102851252A (application number 201210274174.8) discloses an engineered strain of *Gluconobacterium oxysporum* that produces the vitamin C synthesis intermediate sorbitol, along with its construction method and application. Through genetic engineering technology, the strain derived from common keto-producing gulonic acid bacteria (… Ketogulonigenium vulgare ) sorbitol dehydrogenase gene ( sdh ) cloned into glucosinolates ( Gluconobacter oxydans In this study, a strain was obtained that produces sorbitol and sorbitone. G.oxydans The engineered bacteria have laid the foundation for further developing one-step fermentation engineered bacteria to produce the vitamin C precursor 2-KGA.

[0003] Regularly clustered interspaced short palindromic repeats (CRISPR) are widely found in bacteria and are an immune defense system developed over a long period of evolution to resist the invasion of foreign DNA. Because type II CRISPR systems require only the expression of a single protein and have a relatively simple structure, they are most widely used in microorganisms, such as *Escherichia coli*, *Corynebacterium glutamicum*, and *Saccharomyces cerevisiae*. Compared to Cas9 proteins, Cpf1 proteins (Cas12a) have a smaller molecular weight, lower toxicity, and can self-process and modify pre-crRNA without binding tracrRNA, making them suitable for genome editing in specific strains. Cpf1 proteins contain two nuclease domains; site-directed mutagenesis can yield dCpf1 proteins that lose their DNA-cutting ability. These dCpf1 proteins, guided by crRNA, bind to specific sites in the genome to achieve transcriptional repression.

[0004] Currently, CRISPR systems are being used in an increasing number of model and non-model strains, playing a significant role in reshaping metabolic pathways, identifying new and highly efficient metabolic enzymes, and optimizing chassis cells. However, due to the immaturity of the intrinsic genetic manipulation system of ketogenic gulonic acid bacteria and the lack of key regulatory elements such as strong promoters, there are few reports on the application of highly efficient gene editing systems. No such CRISPR system has yet been constructed for gene editing in ketogenic gulonic acid bacteria, severely hindering the genetic modification of the genomic level. Summary of the Invention

[0005] To address the shortcomings of the existing technology, this invention provides a ketogenic gulonic acid bacteria gene inhibition system and its application.

[0006] The technical solution of the present invention is as follows: A ketogenic gulonic acid bacteria gene inhibition system, the system comprising the CRISPRi plasmid, the CRISPRi plasmid comprising a gene encoding dCpf1, crRNA, and a promoter expressing crRNA; the gene encoding dCpf1 is located downstream of promoter P1066, and the crRNA and the promoter expressing crRNA are located upstream of promoter P1066.

[0007] According to a preferred embodiment of the present invention, the nucleotide sequence of the promoter P1066 is shown in SEQ ID NO.6.

[0008] According to a preferred embodiment of the present invention, the gene encoding dCpf1 is derived from the novel culprit, *Francis*. Francisella novicida It is derived from the Cpf1 encoding gene by a mutation at the D917A site. The nucleotide sequence of the gene encoding dCpf1 is shown in SEQ ID NO.1.

[0009] According to a preferred embodiment of the present invention, the promoter for expressing crRNA includes pKan, the nucleotide sequence of which is shown in SEQ ID NO.3.

[0010] The application of the above system in gene editing of ketogenic gulonic acid bacteria.

[0011] According to a preferred embodiment of the present invention, the application involves constructing a crRNA designed based on the target gene sequence into the above-mentioned system, and then transferring it into ketogenic gulonic acid bacteria to obtain a gene-suppressing strain.

[0012] According to a preferred embodiment of the present invention, the ketogenic gulonic acid bacteria includes ketogenic gulonic acid bacteria WTF0114 with accession number CCTCC NO: M2024406.

[0013] Microbial cells containing the above-mentioned systems.

[0014] According to a preferred embodiment of the present invention, the microbial cell is a ketogenic gulonic acid bacterium.

[0015] The application of microbial cells containing the above system in the preparation of products containing 2-keto-L-gulonic acid, wherein the microbial cells are keto-gulonic acid-producing bacteria.

[0016] The above system was applied to the study of the biological functions of specific genes in ketogulonic acid bacteria.

[0017] Beneficial effects: This invention addresses the problems of low gene editing efficiency and poor applicability in keto-producing gulonic acid bacteria. Due to the unique genetic background of keto-producing gulonic acid bacteria, their growth ability is weak, and the CRISPR Cas9 system has high toxicity, thus failing to achieve precise and efficient gene editing. Furthermore, the inventors discovered through experiments that the λ-Red homologous recombination system easily damages the bacterial cells, and the strains themselves cannot repair the damage, thus failing to obtain positive recombinant colonies, making it unsuitable for keto-producing gulonic acid bacteria. This invention utilizes the CRISPR Cas12-related dCpf1 protein to construct a gene repression system, providing a new method for inhibiting target genes in keto-producing gulonic acid bacteria. This method can rapidly and effectively inhibit the expression of target genes, improving the gene editing efficiency of keto-producing gulonic acid bacteria.

[0018] After numerous experiments, the inventors screened out the endogenous promoter P1066 of ketogenic gulonic acid bacteria. This promoter can stably and efficiently transcribe in the strain and accurately drive the expression of the dCpf1 encoded gene, solving the problems of poor adaptability and high off-target rate of exogenous promoters, and ensuring the specificity and reliability of gene repression. Attached Figure Description

[0019] Figure 1 A bar chart characterizing the fluorescence intensity of the endogenous promoter P1066; Figure 2 Map of recombinant plasmid pMCS2-P1066-dCpf1(D917A)-pKan-crRNApgmB; Figure 3 To suppress pgmB A bar chart showing the relative transcriptional levels before and after gene generation; Figure 4 A bar chart characterizing the fluorescence intensity of different endogenous promoters; Figure 5 The graph shows the changes in D-sorbitol during the fermentation process of recombinant ketogenic gulonic acid bacteria; where M represents the original strain, D represents ketogenic gulonic acid bacteria WTF0114 / pMCS2-P1066-dCpf1(D917A)-pKan-crRNApgmB, E represents ketogenic gulonic acid bacteria WTF0114 / pMCS2-P1066-dCpf1(E1006A)-pKan-crRNApgmB, and DE represents ketogenic gulonic acid bacteria WTF0114 / pMCS2-P1066-dCpf1(D917A and E1006A)-pKan-crRNApgmB. Detailed Implementation

[0020] The technical solution of the present invention will be further described below with reference to the embodiments, but the scope of protection of the present invention is not limited thereto.

[0021] Unless otherwise specified, the drugs and reagents used in the examples are common products already on the market. All contents not described in detail in the examples are based on the prior art.

[0022] In the following examples, the ketogenic gulonic acid bacterium WTF0114 used is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M2024406. This strain has been disclosed in Chinese patent document CN119061081A (application number 202411197796.4), and this application does not involve the deposit of the patent strain.

[0023] The culture media and detection methods involved in the following examples are as follows: 1. Culture medium HJ medium: yeast extract 5.0 g / L, peptone 15.0 g / L, urea 5.0 g / L, anhydrous magnesium sulfate 2.5 g / L, sorbitol 15.0 g / L, solvent: water, pH 7.0.

[0024] Liquid sorbitol medium: sorbitol 20 g / L, peptone 10 g / L, yeast extract 3 g / L, beef extract 3 g / L, corn steep liquor 3 g / L, urea 1 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate 0.2 g / L, calcium carbonate 1 g / L, solvent: water, pH 6.4-6.7; solid sorbitol medium is liquid sorbitol medium with 17 g / L agar added.

[0025] Fermentation medium: D-sorbitol 50g / L, yeast extract 2g / L, corn steep liquor 8g / L, urea 8g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate 0.2g / L, calcium carbonate 1g / L, solvent: water, pH 6.7.

[0026] 2. Preparation of competent cells of ketogenic gulonic acid bacteria: Pick a single colony and inoculate it into liquid sorbitol medium and culture overnight at 30°C. The next day, transfer it to 100 mL of liquid sorbitol medium at a volume ratio of 10% and culture at 30°C for 8-10 h. Take 25 mL of fermentation broth, centrifuge and discard the supernatant, add HEPES buffer and wash twice, centrifuge at 4°C and discard the supernatant, wash the cells twice with pre-cooled 10% glycerol and resuspend them in 1 mL, and dispense 90 μL each time.

[0027] 3. Keto-based gulonic acid bacteria transformation: Add 2 μg of plasmid to 90 μL of competent keto-based gulonic acid bacteria cells, mix well, and then transfer to a pre-cooled electroporation cuvette with a 2 mm gap. Perform electroporation using a Bio-Rad MicroPulser instrument at 2.5 kV for 5 ms. Immediately after electroporation, add 1 mL of HJ medium and incubate at 30°C for 4 h. Then, plate the mixture onto solid sorbitol agar plates containing 25 μg / mL kanamycin and incubate for approximately 72 h until visible colonies appear.

[0028] 4. qRT-PCR Procedure: Single colonies of wild-type keto-producing gulonic acid bacteria WTF0114 and recombinant keto-producing gulonic acid bacteria WTF0114 / pMCS2-P1066(D917A)-dCpf1-pKan-crRNApgmB were inoculated into 100 mL of liquid sorbitol medium and incubated at 30°C for 30 h. Then, they were transferred to 100 mL of fermentation medium at a volume ratio of 10% and incubated at 30°C for an additional 30 h. Fermentation broth samples were centrifuged after 12 h to obtain bacterial cells. Total RNA was extracted from the samples using a bacterial RNA kit from Omega (Guangzhou, China) according to the manufacturer's instructions, and the concentration was measured using an Eppendorf spectrophotometer (Hamburg, Germany).

[0029] cDNA was synthesized from total RNA using the PrimeScript™ RT Master Mix (Perfect Real Time) kit from Takara (Dalian, China) according to the manufacturer's instructions. The 16S rRNA gene was used as an internal standard for assay. pgmB Gene expression levels. qRT-PCR analysis was performed on a LightCycler 480II instrument (Roche, Mannheim, Germany). The reaction mixture was prepared and the amplification program was set according to the instructions for the Takara Premix Ex Taq II (TliRNaseH Plus) kit. CT values ​​were determined using LightCycler software (version 3.3), with each sample analyzed in triplicate.

[0030] The qRT-PCR primer sequences are as follows: 16S rRNA-F: 5'-ACCCTTGTCTTTAGTTTGCCAGCAC-3'; 16S rRNA-R: 5'-CCACTGTCACCGCCATTGTAGC-3'; pgmB -F: 5'-CGAGGCGCTGGCCGGCGTCCATG-3'; pgmB -R: 5'-GCGCATGAGGTGCTGACGGCGCT-3'.

[0031] 5. Fermentation steps: Select a single colony and inoculate it into liquid sorbitol medium. After culturing at 30°C for 30 hours, transfer it to 100 mL of fermentation medium at a volume ratio of 10%. Cultivate at 30°C and 200 rpm for 120 hours. Store the fermentation broth sample in a -20°C refrigerator.

[0032] 6. Detection Method for Key Components in Fermentation Broth: The content of D-sorbitol in the fermentation broth was determined by high-performance liquid chromatography (HPLC). Chromatographic column: Aminex HPX-87H; mobile phase: 3mM sulfuric acid; column temperature: 35℃; flow rate: 0.2mL / min; injection volume: 20 μL. Before detection, the fermentation broth sample was removed from the freezer and thawed at room temperature. It was centrifuged at 12000 rpm for 10 min to remove bacterial cells. The sample was diluted with the mobile phase until the concentration of the analyte was within the detection range of the standard curve. The concentration of D-sorbitol was then determined by HPLC.

[0033] Example 1 Construction of the recombinant vector pMCS2-P1066-dCpf1(D917A) ① Amplification and functional verification of endogenous promoter P1066 in ketogenic gulonic acid bacteria WTF0114 Design primers F-MCS2 and R-MCS2 for linearization of plasmid pBBR1-MCS2 F-MCS2: 5'-CGCATCCTCACGATAATATCCGGGTAGG-3'; R-MCS2: 5'-CTCTGAATGGCGGGAGTATGAAAAGTATGGCTGAAGCGC-3'.

[0034] Using commercially available plasmid pBBR1-MCS2 as a template, PCR linearization amplification was performed using primers F-MCS2 and R-MCS2. 2×Phanta Max Master Mix (Vazyme) high-fidelity DNA polymerase was selected. The amplification conditions were: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 15 s, 64℃ annealing for 15 s, 72℃ extension for 3 min, 30 cycles; and 72℃ final extension for 5 min.

[0035] Primers FG and RG were designed for amplifying the GFP gene: FG: 5'-CTTTTCATACTCCCGCCATTCAGAGATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCC-3'; RG: 5'-GGATATTATCGTGAGGATGCGTTATTTGTAGAGCTCATCCATGCC-3'.

[0036] Using commercially available plasmid pET28a-EGFP as a template, the GFP gene was amplified using primers FG and RG. 2×Phanta Max Master Mix high-fidelity DNA polymerase was selected, and the amplification conditions were: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 15 s, 64℃ annealing for 15 s, 72℃ extension for 30 s, 30 cycles; 72℃ final extension for 5 min.

[0037] The PCR products from the two steps described above were purified and recovered. The purified fragments were then recombined into the vector pMCS2-GFP using an Infusion-Cloning kit (Vazyme), and transformed into *E. coli* DH5α competent cells. The transformants were sent to Beijing Qingke for sequencing. Those correctly sequenced were considered positive transformants, and the plasmid pMCS2-GFP (nucleotide sequence shown in SEQ ID NO. 5) was extracted from the positive transformants.

[0038] Design primers F-1 and R-1 for linearization of plasmid pMCS2-GFP: F-1: 5'-ATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCC-3'; R-1: 5'-CTCTGAATGGCGGGAGTATGAAAAGTATGGC-3'.

[0039] Using the plasmid pMCS2-GFP obtained above as a template, PCR linearization amplification was performed using primers F-1 and R-1. 2×Phanta Max Master Mix high-fidelity DNA polymerase was selected, and the amplification conditions were: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 4 min, 30 cycles; 72℃ final extension for 5 min.

[0040] Design primers F-2 and R-2 for amplifying the endogenous promoter P1066 (nucleotide sequence as shown in SEQ ID NO. 6): F-2: 5'-CTTTTCATACTCCCGCCATTCAGAGGGAACGGATCATGTTCGCAATTCCCGGCAAGCAAGCCG-3'; R-2: 5'-GTGAAAAGTTCTTCTCCTTTACTCATAAGCCCCTCCGATATGTCTTTGGGTCGTCTAACAGCAGCG-3'.

[0041] Using the genome of *Gastroenterobacter ketogenes* WTF0114 as a template, PCR amplification of promoter P1066 was performed using primers F-2 and R-2. 2×PhantaMax Master Mix high-fidelity DNA polymerase was selected. The amplification conditions were: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 15 s, 70℃ annealing for 15 s, 72℃ extension for 30 s, 30 cycles; and 72℃ final extension for 5 min.

[0042] The PCR products from the two steps described above were purified and recovered. Using an Infusion-Cloning kit (Vazyme), the purified fragments were recombined into the vector pMCS2-P1066-GFP, which was then transformed into *E. coli* DH5α competent cells. Transformants were sent to Beijing Qingke for sequencing; those correctly sequenced were considered positive transformants. The plasmid pMCS2-P1066-GFP was extracted from the positive transformants and transformed into *Bacillus ketogenes* WTF0114 competent cells. After colony PCR verification, recombinant *Bacillus ketogenes* WTF0114 / pMCS2-P1066-GFP was obtained.

[0043] Ketogenic gulonic acid bacteria WTF0114 was used as the control group, and recombinant ketogenic gulonic acid bacteria WTF0114 / pMCS2-P1066-GFP was used as the experimental group. Single colonies were picked and inoculated into 100 mL of liquid sorbitol medium and cultured at 30 °C for 30 h. Then, they were transferred to 100 mL of HJ medium at a volume ratio of 10% and cultured at 30 °C for a further period. Samples were taken after 12 h of culture, and the bacterial cells were collected by centrifugation at 3000 rpm. The bacterial cells were washed twice with PBS buffer (pH 7.0) and resuspended in an equal volume. The fluorescence intensity of the samples was measured using a microplate reader under conditions of excitation wavelength of 485 nm and emission wavelength of 533 nm.

[0044] The results are as follows Figure 1 As shown, the fluorescence intensity of the experimental group was 5 times that of the control group, indicating that promoter P1066 can be transcribed normally in ketogenic gulonic acid bacteria.

[0045] ② Construction of the recombinant vector pMCS2-P1066-dCpf1 (D917A) Design primers F-6 and R-6 for linearization of plasmid pMCS2-P1066-GFP: F-6: 5'-CGCATCCTCACGATAATATCCGGGTAG-3'; R-6: 5'-AAGCCCCTCCGATATGTCTTTGGG-3'.

[0046] Using the plasmid pMCS2-P1066-GFP (nucleotide sequence shown in SEQ ID NO.7) constructed in step ① as a template, PCR linearization amplification was performed using primers F-6 and R-6. 2×Phanta Max Master Mix high-fidelity DNA polymerase was selected, and the amplification conditions were: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 15 s, 56℃ annealing for 15 s, 72℃ extension for 3 min, 30 cycles; 72℃ final extension for 5 min.

[0047] Design primers F-7 and R-7 for amplifying the dCpf1 (D917A) sequence: F-7: 5'-CAAAGACATATCGGAGGGGCTTATGTCAATTTATCAAGAATTTG-3'; R-7: 5'-GGATATTATCGTGAGGATGCGTTAGTTATTCCTATTCTGCACGAACTC-3'.

[0048] Using the commercially available plasmid pLcx-dCpf1 (purchased from Molecular Cloud, nucleotide sequence shown in SEQ ID NO.8) as a template, the mutation site of this plasmid is at position 917 (D917A). PCR amplification of dCpf1 (D917A) was performed using primers F-7 and R-7. 2×Phanta Max Master Mix high-fidelity DNA polymerase was selected. The amplification conditions were: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 15 s, 50℃ annealing for 15 s, 72℃ extension for 2 min, 30 cycles; 72℃ final extension for 5 min.

[0049] The PCR products from the above two steps were purified and recovered. The purified fragments were recombined into the vector pMCS2-P1066-dCpf1(D917A) using an Infusion-Cloning kit and transformed into E. coli DH5α competent cells. The transformants were sent to Beijing Qingke for sequencing. The correctly sequenced vector was pMCS2-P1066-dCpf1(D917A) (nucleotide sequence as shown in SEQ ID NO.2).

[0050] Example 2 Construction of the CRISPRi system for inhibiting the gene production of keto-producing gulonic acid bacteria and its application in inhibiting gene production in the genome of keto-producing gulonic acid bacteria WTF0114. pgmB The gene (nucleotide sequence shown in SEQ ID NO.4) is suppressed.

[0051] Primers F-8 and R-8 were designed for linearization of the recombinant vector pMCS2-P1066-dCpf1 (D917A) constructed in Example 1: F-8: 5'-CGGCTATGACTGGGCACAACAAATAATTTCTACTGTTGTAGATCGAGGCGCTGGCCGGCGTCCATG-3'; R-8: 5'-CGGCAGAACGCCTCGGGCTGAAGAAATAATTTCTACTGTTGTAGATCGCATCCTCACGATAATATCCGGGTAGGCGCAATC-3'.

[0052] Using the recombinant vector pMCS2-P1066-dCpf1 (D917A) as a template, PCR linearization amplification was performed using primers F-8 and R-8. 2×Phanta Max Master Mix high-fidelity DNA polymerase was selected. The amplification conditions were: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 15 s, 59℃ annealing for 15 s, 72℃ extension for 5 min, 30 cycles; 72℃ final extension for 5 min.

[0053] Design primers F-9 and R-9 for amplifying the crRNA pgmB sequence: F-9: 5'-AAATAATTTCTACTGTTGTAGATCGAGGCGCTGGCCGGCGTCCATG-3'; R-9: 5'-CGGCAGAACGCCTCGGGCTGAAGAAATAATTTCTACTGTTGTAGAT-3'.

[0054] The crRNA pgmB sequence (nucleotide sequence shown in SEQ ID NO. 9, containing the pKan promoter, whose nucleotide sequence is shown in SEQ ID NO. 3) was designed according to crRNA design principles. Gene synthesis was performed by Beijing Qingke. Design principles: using the crRNA design website http: / / www.rgenome.net / , the species FnCpf1 was selected in Cas-Designer. Francisella novicida PAM type: 5'-TTN-3', input the target gene sequence into QuerySequence; select RGENTarget sequences with an Out-of-frame Score greater than 66 and a GC content of 40%-60% from the 20nt results of the obtained crRNAs, and ensure that they do not contain consecutive TTTT sequences.

[0055] Using the synthesized crRNApgmB sequence as a template, PCR amplification was performed using primers F-9 and R-9. 2×Phanta Max Master Mix high-fidelity DNA polymerase was selected, and the amplification conditions were: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 5 s, 67℃ annealing for 15 s, 72℃ extension for 10 s, 30 cycles; 72℃ final extension for 5 min.

[0056] The PCR products from the two steps described above were purified and recovered. Using an infusion-cloning kit, the purified fragments were recombined into the vector pMCS2-P1066-dCpf1(D917A)-pKan-crRNApgmB, which was then transformed into *E. coli* DH5α competent cells. The transformants were sent to Beijing Qingke for sequencing. Sequencing confirmed the recombinant plasmid pMCS2-P1066-dCpf1(D917A)-pKan-crRNApgmB, i.e., *Ketotrophoblastic gulonic acid bacteria*. pgmB Gene repression system, plasmid map as follows Figure 2 As shown.

[0057] The recombinant plasmid was transformed into competent cells of ketogenic gulonic acid bacteria WTF0114. After colony PCR verification, the recombinant ketogenic gulonic acid bacteria WTF0114 / pMCS2-P1066-dCpf1(D917A)-pKan-crRNApgmB was obtained.

[0058] Using strain WTF0114 as the control group and recombinant ketogenic gulonic acid bacterium WTF0114 / pMCS2-P1066-dCpf1(D917A)-pKan-crRNApgmB as the experimental group, qRT-PCR analysis was performed.

[0059] The results are as follows Figure 3 As shown, compared with the control bacteria, the recombinant keto-producing gulonic acid bacteria WTF0114 / pMCS2-P1066-dCpf1(D917A)-pKan-crRNApgmB contained... pgmB A 30% reduction in gene expression indicates that this inhibitory system is effective against gene expression. pgmB Gene expression has an inhibitory effect and can be used to suppress genes in ketogenic gulonic acid bacteria.

[0060] Comparative Example 1 Comparison of endogenous promoter strength of ketogenic gulonic acid bacteria Primers F-829 and R-829 were designed for amplifying the endogenous promoter P829 (nucleotide sequence as shown in SEQ ID NO. 10), primers F-866 and R-866 were designed for amplifying the endogenous promoter P866 (nucleotide sequence as shown in SEQ ID NO. 11), and primers F-1457 and R-1457 were designed for amplifying the endogenous promoter P1457 (nucleotide sequence as shown in SEQ ID NO. 12). F-829: 5'-CATACTCCCGCCATTCAGAGCCGACCCGCTGATCAATGGCT-3'; R-829: 5'-GTTCTCTCCTTTACTCATTTTTCTGCCCAATGAATTGTCGCGCCGCGC-3'; F-866: 5'-CATACTCCCGCCATTCAGAGCTGGCACCAGATATGCATCGTGGCCG-3'; R-866: 5'-GAAAAGTTCTTCTCCTTTACTCATATTCAACCTGCAGCGAATGGGTTACGCCGCCCTTTCTGAG-3'; F-1457: 5'-CATACTCCCGCCATTCAGAGCTTGGCTGGCTGCAATCGAATATGACACC-3'; R-1457: 5'-GAAAAGTTCTTCTCCTTTACTCATAGAGGGCCTCCTTCATTCGCCCCTATCTTAAC-3'.

[0061] Recombinant ketogenic gulonic acid bacteria WTF0114 / pMCS2-P829-GFP, WTF0114 / pMCS2-P866-GFP, and WTF0114 / pMCS2-P1457-GFP were obtained according to step ① in Example 1. Using ketogenic gulonic acid bacteria WTF0114 as a control, single colonies were picked and inoculated into 100 mL of liquid sorbitol medium. After incubation at 30°C for 30 h, the colonies were transferred to 100 mL of HJ medium at a volume ratio of 10% and incubated at 30°C for an additional 12 h. Samples were taken after centrifugation at 3000 rpm, and the bacterial cells were collected. The cells were washed twice with PBS buffer (pH 7.0) and resuspended in an equal volume. The fluorescence intensity of the samples was measured using a microplate reader under conditions of excitation wavelength of 485 nm and emission wavelength of 533 nm.

[0062] The results are as follows Figure 4As shown, among the four endogenous promoters, P1066 exhibits significantly higher fluorescence intensity than the other promoters, making it a stronger endogenous promoter in this ketogenic gulonic acid bacterium.

[0063] Comparative Example 2 Comparison of the inhibitory effects of different dCpf1 mutants on gene expression in ketogenic gulonic acid bacteria ① Construction of recombinant vectors pMCS2-P1066-dCpf1 (E1006A) and pMCS2-P1066-dCpf1 (D917A and E1006A) Using the commercially available plasmid pHT-XCR6 as a template, the Cpf1 gene was amplified using primers F-7 and R-7, and the recombinant vector pMCS2-P1066-Cpf1 was constructed, following the same steps as in Example 1.

[0064] Design primers FE and RE for dCpf1 (E1006A) and dCpf1 (D917A and E1006A) mutants: FE: 5'-AGGATTTAAATTTTGGATTTAAAAGAGGGCG-3'; RE:5'-CAAAAACCACAATAGCATTATACTC-3'.

[0065] Using the recombinant vector pMCS2-P1066-Cpf1 constructed above and the recombinant vector pMCS2-P1066-dCpf1 (D917A) constructed in Example 1 as templates, PCR linearization amplification was performed using primers FE and RE, respectively. 2×Phanta Max Master Mix high-fidelity DNA polymerase was selected, and the amplification conditions were: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 15 s, 56℃ annealing for 15 s, 72℃ extension for 6 min, 30 cycles; 72℃ final extension for 5 min.

[0066] The PCR products from the above steps were purified and recovered. Using an Infusion-Cloning kit, the purified fragments were recombined with the purified linearized plasmid pMCS2-P1066-GFP (obtained using the same method as step ② in Example 1) to form the vectors pMCS2-P1066-dCpf1 (E1006A) and pMCS2-P1066-dCpf1 (D917A and E1006A), respectively. These vectors were then transformed into E. coli DH5α competent cells. The transformants were sent to Beijing Qingke for sequencing to obtain the recombinant vectors pMCS2-P1066-dCpf1 (E1006A) and pMCS2-P1066-dCpf1 (D917A and E1006A), respectively.

[0067] ② Construction and fermentation verification of recombinant bacteria Using the recombinant vectors pMCS2-P1066-dCpf1(E1006A) and pMCS2-P1066-dCpf1(D917A and E1006A) constructed in the above steps as templates, recombinant ketogenic gulonic acid bacteria WTF0114 / pMCS2-P1066-dCpf1(E1006A)-pKan-crRNApgmB and WTF0114 / pMCS2-P1066-dCpf1(D917A and E1006A)-pKan-crRNApgmB were constructed in accordance with the method of Example 2.

[0068] Using strain WTF0114 as the control group, and recombinant ketogenic gulonic acid bacteria WTF0114 / pMCS2-P1066-dCpf1(D917A)-pKan-crRNApgmB, WTF0114 / pMCS2-P1066-dCpf1(E1006A)-pKan-crRNApgmB, and WTF0114 / pMCS2-P1066-dCpf1(D917A and E1006A)-pKan-crRNApgmB as experimental groups, fermentation was carried out respectively. pgmB The gene is responsible for the phosphorylation and transport of D-sorbitol into the cell. pgmB When the gene is suppressed, the D-sorbitol transport rate slows down, thus slowing down the D-sorbitol consumption rate and increasing the extracellular D-sorbitol residue. Therefore, by comparing the D-sorbitol consumption rates of transformants containing different dCpf1 mutants, it is possible to determine which mutant has a higher suppression efficiency.

[0069] Fermentation product results as follows Figure 5 As shown, combining experimental data and mechanism analysis, it can be concluded that recombinant ketogenic gulonic acid bacteria WTF0114 / pMCS2-P1066-dCpf1(D917A)-pKan-crRNApgmB(D) has the effect of... pgmB The gene repression effect was optimal; compared with the original strain (M) and other recombinant strains (E, DE), the consumption rate of D-sorbitol was significantly slower; due to the presence of ketogenic gulonic acid bacteria... pgmB Functional compensatory pathways (such as regulation by other metabolic transport genes and isoenzymes), and the dCpf1-mediated gene repression is only partially weakened rather than completely inactivated, these factors weaken phenotypic differences. At 120 h of fermentation, the D-sorbitol content did not differ significantly, but in the early stages of culture (24 h, 48 h), the D-sorbitol content of group D strains was significantly higher than that of the original strain. This difference is precisely... pgmB The suppression of gene expression and the decrease in substrate transport rate directly demonstrate the effectiveness of the gene suppression system provided by this invention.

[0070] Comparative Example 3 Gene knockout of ketogenic gulonic acid bacteria based on λ-Red homologous recombination system ① Construction of the recombinant vector pMCS2-P1066-Red Primers FR and RR were designed for Red recombinase gene amplification: FR: 5'-CCCAAAGACATATCGGAGGGGCTTATGGATATTAATACTGAAACTGAGATCAAGC-3'; RR: 5'-CCGGATATTATCGTGAGGATGCGTCATCGCCATTGCTCCCCAAATACAAAAC-3'.

[0071] Using commercially available plasmid pKD46 as a template, PCR linearization amplification was performed using primers FR and RR. 2×PhantaMax Master Mix high-fidelity DNA polymerase was selected, and the amplification conditions were as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 15 s, 65℃ annealing for 15 s, 72℃ extension for 2 min, 30 cycles; 72℃ final extension for 5 min.

[0072] The PCR products from the above steps were purified and recovered. The purified fragments were recombined with the purified linearized plasmid pMCS2-P1066-GFP (obtained by the same method as step ② in Example 1) using an Infusion-Cloning kit to form the vector pMCS2-P1066-Red. The vector was then transformed into E. coli DH5α competent cells. The transformants were sent to Beijing Qingke for sequencing to obtain the recombinant vector pMCS2-P1066-Red.

[0073] ② The obtained recombinant vector pMCS2-P1066-Red was transformed into keto-producing gulonic acid bacteria WTF0114 to obtain recombinant keto-producing gulonic acid bacteria WTF0114 / pMCS2-P1066-Red. Target fragment pairs were designed. pgmB Genes are knocked out.

[0074] Target fragment acquisition: Design primers Ff pgmB, Rf pgmB 、Fr pgmB, Rr pgmB Amplification pgmB Based on the upstream and downstream homologous arms of the gene, primers F-CmR and R-CmR were designed to amplify the chloramphenicol resistance gene.

[0075] Ff pgmB :5'-GCGCATCCTCGCCGCAAGATCC-3'; Rf pgmB:5'-GGCAGATCGCGAATTGCCGATGGTCTGGCGCCGTCGGGTTTGATGGGGACGCTGATC-3'; Fr pgmB :5'-CTGCCTCCCAGAGCCTGATAAAAACGGTCCGCATGAGGTGCTGACGGCGCTGAAGGCGGCGGGC-3'; Rr pgmB :5'-CCGTCAAAGCCTTAGCTGTCCGAGCGGC-3'; F-CmR: 5'-GCGCCAGACATCGGCAATTCGCGATCTGCC-3'; R-CmR: 5'-GACCGTTTTTATCAGGCTCTGGGAGGCAG-3'.

[0076] Using the whole genome of ketotrophic gulonic acid bacteria as a template, primer Ff was used. pgmB, Rf pgmB right pgmB PCR amplification was performed on the upstream 500bp of the gene using 2×PhantaMax Master Mix high-fidelity DNA polymerase. The amplification conditions were: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 5 s, 62℃ annealing for 15 s, 72℃ extension for 45 s, for 30 cycles; and 72℃ final extension for 5 min.

[0077] Using the whole genome of ketogenic gulonic acid bacteria as a template, primers Fr were used. pgmB, Rr pgmB right pgmB PCR amplification was performed on the 500bp downstream of the gene using 2×PhantaMax Master Mix high-fidelity DNA polymerase. The amplification conditions were: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 5 s, 75℃ annealing for 15 s, 72℃ extension for 45 s, for 30 cycles; and 72℃ final extension for 5 min.

[0078] Using commercially available plasmid pCP20 as a template, primer F-CmR was used. 、 R-CmR was used to amplify the chloramphenicol resistance gene by PCR. 2×PhantaMax Master Mix high-fidelity DNA polymerase was selected. The amplification conditions were: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 5 s, 70℃ annealing for 15 s, 72℃ extension for 60 s, 30 cycles; 72℃ final extension for 5 min.

[0079] The PCR products from the above steps were purified and recovered, and then mixed in equal amounts (molar ratio 1:1:1) for fusion PCR amplification to obtain the amplification template. 2×PhantaMax Master Mix high-fidelity DNA polymerase was selected, and the amplification conditions were: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 5 s, 65℃ annealing for 15 s, 72℃ extension for 60 s, 30 cycles; 72℃ final extension for 5 min.

[0080] Using the PCR amplification template obtained in the above steps as a template, and using primer Ff pgmB, Rr pgmB PCR amplification was performed using 2×PhantaMax Master Mix high-fidelity DNA polymerase. The amplification conditions were: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 5 s, 65℃ annealing for 15 s, 72℃ extension for 60 s, for 30 cycles; and a final extension at 72℃ for 5 min. The targeted fragment was obtained after gel recovery, and its nucleotide sequence is shown in SEQ ID NO.13.

[0081] Recombinant ketogenic gulonic acid bacteria WTF0114 / pMCS2-P1066-Red competent cells were prepared, and the target fragment was electroporated into the competent cells. The electroporated bacterial culture was spread on solid sorbitol medium containing 10 μg / mL chloramphenicol and cultured at 30℃ for 72 h.

[0082] Experimental results showed that no colonies grew on the culture medium, indicating that the λ-Red homologous recombination system cannot be used for *Bacillus ketogenes* WTF0114. pgmB Gene knockout.

Claims

1. A ketogenic gulonic acid bacteria gene inhibition system, characterized in that, The system includes the CRISPRi plasmid, which includes a gene encoding dCpf1, crRNA, and a promoter expressing the crRNA; the gene encoding dCpf1 is located downstream of promoter P1066, and the crRNA and the promoter expressing the crRNA are located upstream of promoter P1066.

2. The system as described in claim 1, characterized in that, The nucleotide sequence of the promoter P1066 is shown in SEQ ID NO.

6.

3. The system as described in claim 1, characterized in that, The gene encoding dCpf1 was developed by the novel culprit, *Francis*. Francisella novicida It is derived from the Cpf1 encoding gene by a mutation at the D917A site. The nucleotide sequence of the gene encoding dCpf1 is shown in SEQ ID NO.

1.

4. The system as described in claim 1, characterized in that, The promoter for expressing crRNA includes pKan, the nucleotide sequence of which is shown in SEQ ID NO.

3.

5. The application of the system according to claim 1 in gene editing of ketogenic gulonic acid bacteria.

6. The application as described in claim 5, characterized in that, The application involves constructing crRNA designed based on the target gene sequence into the system described in claim 1, and then transferring it into ketogenic gulonic acid bacteria to obtain a gene-suppressing strain.

7. The application as described in claim 5, characterized in that, The ketogenic gulonic acid bacteria include ketogenic gulonic acid bacteria WTF0114 with accession number CCTCCNO:M2024406.

8. Microbial cells containing the system of claim 1; Preferably, the microbial cells are ketogenic gulonic acid bacteria.

9. The use of microbial cells containing the system of claim 1 in the preparation of products containing 2-keto-L-gulonic acid, wherein the microbial cells are ketogulonic acid-producing bacteria.

10. The application of the system of claim 1 in studying the biological function of specific genes in ketogulonic acid bacteria.

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