An engineered bacillus subtilis, preparation method and application thereof

Abstract

The present application belongs to the technical field of genetic engineering, and particularly relates to an engineered bacillus subtilis, a preparation method and application thereof. In the present application, in order to improve the supply of precursor cytidine triphosphate (CTP) in the process of fermentation of bacillus subtilis for producing cytidine, first, the feedback-inhibited CTP synthetase PyrG E156K is overexpressed to promote the conversion of uridine triphosphate to CTP; then, the repressor gene of the pyrimidine operon pyrR is knocked out to promote the synthesis of uridine monophosphate, and further promote the synthesis of CTP; finally, the pyrimidine nucleotide degradation genes pdp and cdd are knocked out to block the conversion of uridine / cytidine to uracil / cytosine and the conversion of cytidine to uridine, respectively, reduce the consumption of pyrimidine nucleotides, and further facilitate the intracellular accumulation of CTP. The yield of cytidine of the finally constructed engineering bacteria is about 12.6 times that of the original starting bacteria, and the results of the present application further prove the importance of CTP for cytidine synthesis.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to an engineered Bacillus subtilis, a preparation method and application thereof. BACKGROUND

[0002] Cytidine diphosphate-choline, also known as cytidine-5'-diphosphate-choline, is a key intermediate in the synthesis pathway of phosphatidylcholine in organisms, and is crucial for maintaining the structural integrity and function of neuron membranes. As a precursor for the synthesis of neurotransmitter acetylcholine, the large-scale production of cytidine diphosphate-choline is of great importance.

[0003] At present, there are many reports on fermentation production strains of cytidine diphosphate-choline. For example, patents CN115896211A and CN116144559A disclose recombinant engineering bacteria obtained by transforming Escherichia coli as an initial strain, and patent CN116790466A discloses a fermentation strain for preparing cytidine diphosphate-choline by transforming Bacillus subtilis. However, the above-mentioned genetically recombinant strains still have problems such as low fermentation yield of cytidine diphosphate-choline and high cost of fermentation raw materials, which seriously limit the industrial production scale of cytidine diphosphate-choline.

[0004] Among them, phosphocholine cytidyltransferase (CCT) catalyzes the reaction of phosphocholine and cytidine triphosphate (CTP) to generate cytidine diphosphate-choline, therefore, CTP is one of the important precursors for the synthesis of cytidine diphosphate-choline. In microorganisms, uridine triphosphate (UTP) is ammoniated to generate CTP, and uridine monophosphate (UMP) is a precursor for the synthesis of UTP, and CTP is also converted into cytidine monophosphate (CMP), and UMP, CMP, etc. are converted into uridine, cytidine, further into uracil, cytosine and other components, thereby affecting the intracellular accumulation of CTP.

[0005] Therefore, it is of great significance to improve the fermentation effect of cytidine diphosphate-choline of Bacillus subtilis by modifying the related genes of CTP precursors supply of Bacillus subtilis to ensure the energy supply in the fermentation process and avoid the additional loss of carbon source. SUMMARY

[0006] In order to solve the above technical problems, the present application provides an engineered Bacillus subtilis, a preparation method and application thereof.

[0007] The first aspect of the present application provides an engineered Bacillus subtilis, which is achieved by at least one modification of (a1)-(a4) to Bacillus subtilis BSC1-4:

[0008] (a1) introducing or overexpressing a gene encoding a feedback-inhibited cytidine triphosphate synthetase, the gene being pyrGA gene mutant, the encoded PyrG protein of which has glutamic acid at position 156 replaced by lysine;

[0009] (a2) inactivating or knocking out a gene encoding a pyrimidine nucleotide operon repressor pyrR ;

[0010] (a3) inactivating or knocking out a gene encoding a pyrimidine nucleotide phosphatase pdp ;

[0011] (a4) inactivating or knocking out a gene encoding a cytidine deaminase cdd .

[0012] As preferred, before the modification of Bacillus subtilis BSC1-4, the bypass metabolic pathway of choline chloride of Bacillus subtilis BSC1-4 needs to be blocked, and at the same time, the genes opuD and the transcriptional repressor gene opcR are overexpressed to strengthen the utilization and absorption of choline chloride.

[0013] The Bacillus subtilis BSC1-4 is a strain disclosed in the patent CN116790466B of the inventor.

[0014] As further preferred, the method for blocking the bypass metabolic pathway of choline chloride of Bacillus subtilis is to inactivate or knock out the genes gbsB encoding choline dehydrogenase and the genes gbsA encoding glycine betaine aldehyde dehydrogenase.

[0015] The second aspect of the present application is to provide the use of the engineered Bacillus subtilis in the fermentation production of cytidine diphosphate choline.

[0016] Further, the present application also provides a method for the fermentation production of cytidine diphosphate choline by the engineered Bacillus subtilis, comprising the following steps:

[0017] S1 Activation of the strain: the strain stored in a glycerol tube is streaked on an LB agar plate for activation;

[0018] S2 Preparation of the seed liquid: a single colony is picked from the plate of S1 and inoculated into an LB liquid medium, and then cultured at 35-40℃ and 150-220 rpm for 10-18 h to prepare a seed liquid;

[0019] S3 Fermentation culture: the seed liquid in S2 is transferred into a fermentation medium for fermentation culture, and after 3-6 h of fermentation, choline chloride with a final concentration of 0.8-1.5 g / L is added, and the fermentation is continued, and the total fermentation time is 24-30 h to obtain a fermentation broth;

[0020] S4: Collecting of cytidine triphosphate: Take the fermentation broth in S3, add deionized water, mix, and then place in an ice water bath. Use an ultrasonic cell disruptor to break the cells. Perform a first centrifugation, take the supernatant, heat treat the supernatant at 80-100℃ for 3-8 min, then perform a second centrifugation, and take the supernatant to obtain cytidine triphosphate.

[0021] In the above method, as preferred, the components of the LB medium in S1 are: 6-15 g / L of tryptone, 3-8 g / L of yeast extract, and 5-20 g / L of NaCl.

[0022] As preferred, the components of the shake flask fermentation medium in S2 are: 30-50 g / L of glucose, 6-25 g / L of corn syrup dry powder, 8-12 g / L of tryptone, 3-6 g / L of yeast extract, 6-12 g / L of NaCl, 8-15 g / L of (NH4)2SO4, 1-5 g / L of KH2PO4, 5-10 g / L of K2HPO4, and 0.5-2 g / L of MgSO4·7H2O, and the pH is 7.0-7.5.

[0023] As preferred, the inoculation amount of the seed liquid in S3 is 0.5%-1.2% (v / v).

[0024] As preferred, in S4, the first centrifugation is performed at 6000-8000 rpm for 8-15 min, and the second centrifugation is performed at 10000-15000 rpm for 8-12 min.

[0025] The present application has the following beneficial effects:

[0026] The present application introduces or overexpresses a gene encoding a feedback-inhibited cytidine triphosphate synthetase, and modifies the pyrimidine nucleotide synthesis and degradation pathways, thereby reducing the consumption of pyrimidine nucleotides, blocking the conversion of uridine / cytidine to uracil / cytosine and the conversion of cytidine to uridine, promoting the de novo continuous synthesis of UMP, and further promoting the intracellular accumulation of CTP, thereby further strengthening the utilization of CTP in the fermentation production of cytidine triphosphate by the engineered bacteria. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 The metabolic pathway and systematic modification strategy for the production of cytidine triphosphate by the engineered bacteria provided in the present application are shown in the following schematic diagram:

[0028] OpuB, OpuC, OpuD: choline transport proteins; OpcR: transcriptional repressor of choline transport proteins OpuB and OpuC; CKI : encoding choline kinase; CCT: encoding phosphocholine cytidylyltransferase; UMP: uridine monophosphate; UTP: uridine triphosphate; CTP: cytidine triphosphate; CMP: 5-cytidine monophosphate; PPi: pyrophosphate; PyrR: transcriptional repressor of the pyrimidine nucleotide operon pyr pyrG E156K : encoding CTP synthetase without feedback inhibition pdp : encoding pyrimidine nucleotide phosphorylase cdd : encoding cytidine deaminase DETAILED DESCRIPTION

[0029] In order to enable persons skilled in the art to better understand the present application, the present application will be further described in conjunction with specific embodiments.

[0030] I. Preparation and source of strains, plasmids and culture media involved in the present application.

[0031] The information of all strains and plasmids involved in the present application is shown in Table 1, and the primers are synthesized by Genescript Biotech Co., Ltd.

[0032] LB medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, used for general culture of Bacillus subtilis, solid medium added with 15 g / L agar powder, and 16 μg / mL of neomycin or 8 μg / mL of chloramphenicol was added when necessary.

[0033] Shaking flask fermentation medium: glucose 40 g / L, corn syrup dry powder 10 g / L, tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, (NH4)2SO4 10 g / L, KH2PO4 3 g / L, K2HPO4 8 g / L, MgSO4·7H2O 1 g / L, pH 7.2, after 4 h of fermentation, 1 g / L of choline chloride was added.

[0034] Fed-batch initial culture medium: same as the shaking flask fermentation medium, the feeding medium was composed of 600 g / L of glucose and 20 g / L of MgSO4·7H2O and 30 g / L of choline chloride.

[0035] Table 1 Strains and plasmids involved in the experiment

[0036]

[0037] II. Primers and sequences involved in the present application.

[0038] II. Primers and sequences involved in the present application.

[0039] The primers used for PCR are shown in Table 2. ​

[0040] Table 2 PCR primer sequences

[0041] primer sequence name sequence (5'→3') pksL-U1 SEQ ID No. 1 TGCCTCCTGTAATCTCTGA pksL-U2 SEQ ID No. 2 GCCATCTAAGTGTCCGAAA pksL-D1q SEQ ID No. 3 TTTTCGGACACTTAGATGGCGGCACACAACATTGATGAAT pksL-D2 SEQ ID No. 4 TTGATTTGGCTGACTCTATTG pksL-CR1q SEQ ID No. 5 GCAATAGAGTCAGCCAAATCAATCTTCAACTAAAGCACCCAT CR2 SEQ ID No. 6 TTATTCATTCAGTTTTCGTG pksL-G1q SEQ ID No. 7 GCACGAAAACTGAATGAATAAAGGCTGATTACGCAATGG pksL-G2 SEQ ID No. 8 CTTACCTGCTTCTGCTCTT pksL-U2q SEQ ID No. 9 TCTACTATGTTCTCTCTTCCTTTTGCCATCTAAGTGTCCGAAA pyrG-P1 SEQ ID No. 10 AAAAGGAAGAGAGAACATAGTAG pyrG-2 SEQ ID No. 11 CTTTAAGGAATGGCAGTGATT pyrG-3 SEQ ID No. 12 AATCACTGCCATTCCTTAAAG pyrG-4 SEQ ID No. 13 CACCACTGTTTGTCTATCAC pksL-pD1q SEQ ID No. 14 CGTGATAGACAAACAGTGGTGGGCATACAACATTGATGAAT pyrR-U1 SEQ ID No. 15 CCTCGGACTTATGCTTGG pyrR-U2 SEQ ID No. 16 TCCTGCCAGAGCATAGAG pyrR-D1q SEQ ID No. 17 CCTCTATGCTCTGGCAGGATCAACAATCAGGGGGAAAT pyrR-D2 SEQ ID No. 18 CGCTGAATACTCTTGTGATG pyrR-CR1q SEQ ID No. 19 CCATCACAAGAGTATTCAGCGTCATCAACTAAAGCACCCAT pyrR-G1q SEQ ID No. 20 CGCACGAAAACTGAATGAATAACTATATGAACTGTGAGGTGTC pyrR-G2 SEQ ID No.21 TGCTGAAGGCTGAATGAA pdp-U1 SEQ ID No.22 TGAATGTTCTCTTGCCAATC pdp-U2 SEQ ID No.23 CACCTTAGCCATCGTCAA pdp-D1q SEQ ID No.24 ACTTGACGATGGCTAAGGTGCGGCGAAGGTGATTCATA pdp-D2 SEQ ID No.25 AGTGTATGGATGTGGAGTG pdp-CR1q SEQ ID No.26 GCACTCCACATCCATACACTTCTTCAACTAAAGCACCCAT pdp-G1q SEQ ID No.27 CGCACGAAAACTGAATGAATAACTTGTTGGGGCTCTTGAT pdp-G2 SEQ ID No.28 CCGATGAGGATGTTGTCA cdd-U1 SEQ ID No.29 AATCCTAACCATCCGCTATT cdd-U2 SEQ ID No.30 GTACCGCTATCACTTTATATTTTAC cdd-D1q SEQ ID No.31 TGTAAAATATAAAGTGATAGCGGTACGGAAGAATTATTGCCAGGC cdd-D2 SEQ ID No.32 AACACAAGAAACCCACCTTT cdd-CR1q SEQ ID No.33 AAAGGTGGGTTTCTTGTGTTTCTTCAACTAAAGCAGCCAT cdd-G1q SEQ ID No.34 CGCACGAAAACTGAATGAATAAAAGTGATAGAGGAACCATTA cdd-G2 SEQ ID No.35 TGGCAATAATTCTTCCACAG

[0042] Example 1

[0043] The preparation method of an engineered Bacillus subtilis strain is as follows:

[0044] First, the pathway of choline chloride bypass metabolism in Bacillus subtilis was blocked by knocking out the gene encoding choline dehydrogenase using conventional techniques known to those skilled in the art. gbsB Gene encoding glycine betaine aldehyde dehydrogenase gbsA Genes encoding choline transporter transcriptional repressor proteins opcR Meanwhile, according to the method in patent CN116790466B, overexpression opuD To enhance the absorption of choline chloride, strain BSC5-1 was obtained, which exhibits choline chloride bypass metabolism and enhanced choline chloride absorption.

[0045] Next, build pksL Knockout strains were used as controls: using the BS168N genome as a template, fragment U (SEQ ID No. 36, 1118 bp), fragment D (SEQ ID No. 37, 1004 bp), and fragment G (SEQ ID No. 38, 769 bp) were amplified using primer pairs pksL-U1 / pksL-U2, pksL-D1q / pksL-D2, and pksL-G1q / pksL-G2, respectively; BS168N / Δ yrpCm Using the genome as a template, fragment CR was amplified using primer pair pksL-CR1q / CR2 (as disclosed in patent CN116790466B, the same applies below, and will not be repeated). Then, fragments U, D, CR, and G were spliced ​​into fragment UDCRG (SEQ ID No. 39, 4960 bp), and transformed into competent BSC5-1 cells to obtain... pksL Knockout strain BSC6.

[0046] Build pyrG E156K Overexpression strains: Using the genome of BSC6m as a template, fragment U (SEQ ID No. 40, 1118 bp) containing pksL-U1 / pksL-U2q, pyrG-3 / pyrG-4, and pksL-pD1q / pksL-G2 was amplified using primer pairs pksL-U1 / pksL-U2q, pyrG-3 / pyrG-4, and pksL-pD1q / pksL-G2, respectively. pyrGFragment G2 (SEQ ID No. 41, 1306 bp) and fragment DCRG (SEQ ID No. 42, 3842 bp) of the latter half of the gene sequence; plasmid pUC57-simple-PyrG E156K containing P SB promoter and pyrG Fragment G1 (SEQ ID No. 43, 576 bp) of the former half of the gene sequence (containing mutation E156K), splice fragment U, fragment PG1, fragment G2 and fragment DCRG into fragment UPG1G2DCRG (SEQ ID No. 44, 6842 bp), and transform it into competent cells of BSC5-1 to obtain P SB - pyrG E156K In pksL site-integrated expression of pyrG E156K Recombinant strain BSC7.

[0047] pyrR , pdp, cdd Knockout of the gene, the specific operation is as follows:

[0048] BS168N, respectively, using primer pairs pyrR-U1 / pyrR-U2, pyrR-D1q / pyrR-D2 and pyrR-G1q / pyrR-G2 to amplify fragments U (SEQ ID No. 45, 1258 bp), fragment D (SEQ ID No. 46, 960 bp) and fragment G (SEQ ID No. 47, 820 bp); using the genome of BS168N / Δ yrpCm pyrR-CR1q / CR2 to amplify fragment CR, splice fragment U, fragment D, fragment CR and fragment G into fragment UDCRG (SEQ ID No. 48, 5107 bp), and transform it into competent cells of BSC7 to obtain pyrR Knockout strain BSC8.

[0049] BS168N, respectively, using primer pairs pyrR-U1 / pyrR-U2, pyrR-D1q / pyrR-D2 and pyrR-G1q / pyrR-G2 to amplify fragments U (SEQ ID No. 45, 1258 bp), fragment D (SEQ ID No. 46, 960 bp) and fragment G (SEQ ID No. 47, 820 bp); using the genome of BS168N / Δ yrpCmThe genomic DNA of BS168N was used as a template, and the fragment CR was amplified using the primer pair pdp-CR1q / CR2. The fragments U, D, CR, and G were spliced into the fragment UDCRG (SEQ ID No. 52, 5288 bp), and the fragment UDCRG was transformed into the competent cells of BSC8 to obtain the knockout strain BSC9-5. pdp The knockout strain BSC9-6.

[0050] The genomic DNA of BS168N was used as a template, and the fragments U (SEQ ID No. 53, 1601 bp), D (SEQ ID No. 54, 1099 bp), and G (SEQ ID No. 55, 408 bp) were amplified using the primer pairs cdd-U1 / cdd-U2, cdd-D1q / cdd-D2, and cdd-G1q / cdd-G2, respectively; the genomic DNA of BS168N / Δ yrpCm The genomic DNA of BS168N was used as a template, and the fragment CR was amplified using the primer pair cdd-CR1q / CR2. The fragments U, D, CR, and G were spliced into the fragment UDCRG (SEQ ID No. 56, 5177 bp), and the fragment UDCRG was transformed into the competent cells of BSC8 to obtain the knockout strain BSC9-7. cdd The knockout strain BSC9-7.

[0051] In addition, a double knockout strain in which pdp and cdd are simultaneously knocked out was constructed and named BSC9-8.

[0052] Example 2

[0053] The method for fermenting the various engineered B. subtilis strains prepared in Example 1 to produce cytidine diphosphocholine includes the following steps:

[0054] S1: Activation of the strains: the various engineered B. subtilis strains were streaked on LB agar plates for activation;

[0055] S2: Preparation of the seed solution: single colonies were picked from the plates of S1 and inoculated into test tubes containing 5 mL of LB liquid medium, which were then incubated at 37°C and 200 rpm for 12 h to prepare the seed solution;

[0056] S3: Fermentation: the seed solution of S2 was transferred into 250 mL conical flasks containing 30 mL of fermentation medium at an inoculation amount of 1% (v / v), and then incubated at 37°C and 220 rpm. After 4 h of fermentation, 1 g / L of choline chloride was added, and the fermentation was continued for a total of 24 h to obtain the fermentation broth.

[0057] Collection of S4 citicoline: Take the fermentation broth from S3, add deionized water and mix well. Place it in an ice-water bath and use an ultrasonic cell disruptor to disrupt the cells. Centrifuge at 8000 rpm for 10 min, take the supernatant, heat-treat the supernatant at 100℃ for 5 min, and then centrifuge at 12000 rpm for 10 min. Take the supernatant to obtain the product.

[0058] During fermentation, the OD of the bacterial suspension 600 The value and the total yield of cytidine diphosphate choline in the fermentation broth were determined.

[0059] In this invention, the concentration of cytidine diphosphate choline is determined by high-performance liquid chromatography (HPLC). The specific method is as follows:

[0060] HPLC analysis conditions: LC-2030 high-performance liquid chromatograph (Shimadzu); chromatographic column, PC HILIC column (250 mm × 4.6 mm, 5 μm); column temperature, 30℃; mobile phase A: 0.5% formic acid aqueous solution, mobile phase B: acetonitrile / methanol (8:2). v / v ) solution, A:B in a 4:6 ratio ( v / v Isocratic elution was performed at a ratio of 1:1; flow rate: 1.0 mL / min; detector: UV detector; detection wavelength: 280 nm; injection volume: 20 μL.

[0061] Determination of total cytidine diphosphate choline concentration: Take the supernatant obtained from S4 and dilute it with 20% acetonitrile solution ( v / v After appropriate dilution and filtering through a 0.22 μm filter membrane, HPLC analysis can be performed.

[0062] Using control strain BSC5-1, pksL Knockout strain BSC6 pyrG E156K Overexpression strain BSC7, pyrR Knockout strain BSC8 pdp Knockout strain BSC9-6 cdd Knockout strain BSC9-7 and pdp and cdd Double knockout strain 9-8 was fermented, and the growth of each strain during the fermentation process is shown in Table 3.

[0063] Table 3. Growth of the strains

[0064]

[0065] Note: * The results showed a significant difference compared to the control strain (P < 0.05).** Significant difference (P<0.01) compared with the control strain.

[0066] BSC6 is the control strain of BSC7; BSC7 is the control strain of BSC8; BSC8 is the control strain of BSC9-6, 9-7 and 9-8, see Table 4.

[0067] The results of Table 3 show that overexpression of the CTP synthetase (pyrG) that is not feedback inhibited on the basis of BSC6 pyrG E156K ) leads to significant inhibition of the growth of strain BSC7, however, after further knock-out of the pyrimidine operon repressor gene (pyrR) on the basis of BSC7 pyrR , the growth level of strain BSC8 is effectively restored, and other genetic modifications have no significant effect on the growth of the strain.

[0068] The cytidine phosphate choline production of each strain after fermentation for 12 h and 24 h is shown in Table 4.

[0069] Table 4 Cytidine phosphate choline production of different strains after fermentation for 12 h and 24 h (mg / L)

[0070] Strain 12 h 24 h BSC5-1 757.6±9.9 1181.6±11.2 BSC6 749.3±14.2 1114.6 # ±15.9]]> BSC7 795.7 ## ±6.1]]> 1219.7 ## ±14.3]]> BSC8 819.2 ## ±4.3 1309.5 ## ±3.0]]> BSC9-6 802.0 # ±6.5]]> 1324.6 ## ±1.4]]> BSC9-7 832.5 # ±4.3]]> 1384.9 ## ±5.1]]> BSC9-8 846.9 # ±17.2]]> 1441.4 ## ±15.7]]>

[0071] The results of Table 4 show that each step of genetic modification in the application brings stable improvement in production. Strain BSC7 (overexpression of pyrG) pyrG E156K ) still achieves a 24 h production of 1219.7±14.3 mg / L, which is 9.4% higher than its control strain BSC6, proving the direct effectiveness of strengthening the CTP synthesis step; strain BSC8 (knock-out of pyrR) pyrR ) has further improved production to 1309.5±3.0 mg / L while restoring growth, which is 7.4% higher than BSC7; on the basis of BSC8, by knocking out pdp and cdd genes to block the CTP degradation pathway, the production is continuously improved, among which, double-knockout strain BSC9-8 shows the best performance, with a 24 h production of 1441.4±15.7 mg / L, which is 22.0% higher than strain BSC5-1, and is 12.6 times the production of the original starting strain BSC1-4 (114.3 mg / L in patent CN116790466B).

[0072] The above merely describes preferred specific embodiments of the application, but the protection scope of the application is not limited thereto, and any person skilled in the art can make equivalent substitutions or changes to the technical solutions and inventive concepts of the application within the technical scope disclosed by the application, which should be covered within the protection scope of the application.

Claims

1. An engineered Bacillus subtilis strain, characterized in that, obtained by the following modification to Bacillus subtilis BSC1-4 disclosed in patent CN116790466B: First, knock out the gene gbsB encoding choline dehydrogenase, knock out the gene gbsA encoding glycine betaine aldehyde dehydrogenase, knock out the gene opcR encoding choline transporter transcriptional repressor, and overexpress opuD to obtain a strain BSC5-1 with choline chloride bypass metabolism and choline chloride absorption enhancement; Then, on the basis of the strain BSC5-1, the gene encoding the phosphoglycerate pksL mutase was knocked out, the gene encoding the feedback-resistant phosphoglycerate pyrG mutase was introduced, and the gene encoding the feedback-resistant phosphoglycerate pksL mutase was overexpressed. pyrG E156K The overexpression strain, i.e., the engineered Bacillus subtilis, was named BSC7.

2. The engineered B. subtilis strain of claim 1, wherein, The modification further comprises inactivating or knocking out the gene encoding pyrimidine nucleotide operon repressor on the basis of the engineered Bacillus subtilis BSC7 pyrR to obtain an engineered Bacillus subtilis, designated as BSC8.

3. The engineered B. subtilis strain of claim 2, wherein, On the basis of the engineered Bacillus subtilis BSC8, at least one of the following modifications is further performed: Inactivation or knock-out of a gene encoding a pyrimidine nucleotide phosphorylase pdp ; Inactivation or knock-out of a gene encoding cytidine deaminase cdd .

4. The use of the engineered Bacillus subtilis according to any one of claims 1-3 in the fermentation production of cytidine diphosphate choline.

5. The method for the fermentation production of cytidine 5'- monophosphate choline using the engineered B. subtilis according to any one of claims 1 to 3, characterized in that, The steps include: S1 Activation of the strain: the strain stored in a glycerol tube is streaked on an LB agar plate for activation; S2 Preparation of seed liquid: a single colony is picked from the plate of S1 and inoculated in LB liquid medium, and then cultured at 35-40°C and 150-220 rpm for 10-18 h to prepare seed liquid; S3 Fermentation culture: the seed liquid in S2 is transferred into fermentation medium for fermentation culture, and 0.8-1.5 g / L of choline chloride is added after 3-6 h of fermentation, and then the fermentation is continued, and the total fermentation time is 24-30 h to obtain fermentation liquid; S4 Collection of cytidine diphosphate choline: the fermentation liquid in S3 is mixed with deionized water, and then placed in an ice water bath, and then broken by an ultrasonic cell disruptor, and then centrifuged for the first time, and then the supernatant is collected, and then the supernatant is heated at 80-100°C for 3-8 min, and then centrifuged for the second time, and then the supernatant is collected to obtain cytidine diphosphate choline.

6. The method of claim 5, wherein, The inoculation amount of seed liquid in S3 is 0.5%-1.2%.

7. The method of claim 5, wherein, In S4, the first centrifugation is performed at 6000-8000 rpm for 8-15 min, and the second centrifugation is performed at 10000-15000 rpm for 8-12 min.

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