RECOMBINANT MICROORGANISM FOR PRODUCING CITICOLINE AND METHOD FOR PRODUCING CITICOLINE

Abstract

The present invention provides a recombinant microorganism for producing citicoline and a method for producing citicoline by using the recombinant microorganism, wherein genes for degradation and utilization of citicoline, choline, and phosphocholine are knocked out, In addition, a pyrimidine nucleoside synthesis pathway is genetically engineered to remove feedback inhibition to the synthesis pathway. A yield of more than 20 g/L of citicoline can be obtained with recombinant strains in a 5-liter fermenter by means of a biological fermentation method, achieving industrial mass production with low citicoline production costs and less pollution; therefore, the method is a simple, environmentally friendly and has a relatively high promotion and application value.

Claims

1. A recombinant microorganism for producing citicoline, wherein the recombinant microorganism has one or a plurality of the following features: 1) not producing enzymes involved in reuse of citicoline and/or choline and/or phosphocholine; 2) the activity of choline kinase for catalyzing choline chloride to generate phosphocholine being higher than that of a wild-type microorganism; 3) the activity of cytidylyltransferase for catalyzing phosphocholine to generate citicoline being higher than that of a wild-type microorganism; and 4) the activity of choline transporter protein, for transporting choline chloride into a cell being higher than that of a wild-type microorganism.

2. The recombinant microorganism for producing citicoline according to claim 1, wherein the enzymes involved in reuse of citicoline comprise cytidine-5-diphosphoinositol hydrolase and cytidine-5-diphosphate-diacylglycerol pyrophosphatase; the enzymes involved in reuse of choline comprise choline dehydrogenase; and the enzymes involved in reuse of phosphate choline comprise alkaline phosphatase and acid phosphatase.

3. The recombinant microorganism for producing citicoline according to claim 1, wherein the choline kinase comprises CKI1 or EKI1 derived from Saccharomyces cerevisiae, or LicC derived from Streptococcus.

4. The recombinant microorganism for producing citicoline according to claim 1, wherein the cytidylyltransferase comprises PCT1, CDS1, and ECT1 derived from Saccharomyces cerevisiae; or CdsA, IspD, MocA, KdsB, and Cca derived from Escherichia coli; or LicC derived from Streptococcus; or CD36_40620 derived from Candida dubliniensis.

5. The recombinant microorganism for producing citicoline according to claim 1, wherein the choline transporter protein comprises BetT derived from Escherichia coli.

6. The recombinant microorganism for producing citicoline according to claim 1, wherein the recombinant microorganism comprises Escherichia coli.

7. The recombinant microorganism for producing citicoline according to claim 6, wherein in a pyrimidine synthesis pathway of the Escherichia coli, uridine-5-monophosphate phosphorylase for degrading uridine-5-monophosphate into uridine is not produced; and the uridine-5-monophosphate phosphorylase comprises UmpH, UmpG, PhoA, AphA, and YjjG.

8. The recombinant microorganism for producing citicoline according to claim 6, wherein a repressor protein encoding gene of a pyrimidine synthesis pathway of the recombinant microorganism is knocked out and/or feedback inhibition of a final product in the pyrimidine synthesis pathway is removed.

9. The recombinant microorganism for producing citicoline according to claim 8, wherein a repressor protein encoding gene of the pyrimidine synthesis pathway of the Escherichia coli is knocked out and/or feedback inhibition in the pyrimidine synthesis pathway is removed by means of one or a plurality of the following processes: 1) knocking out an encoding gene of transcription inhibition repressor protein of an encoding gene of carbamoyl phosphate synthetase; 2) expressing a mutant S948F of the carbamoyl phosphate synthetase; 3) knocking out an encoding gene of a pyrI subunit of aspartate carbamyltransferase; 4) expressing phosphoribosylpyrophosphate kinase or a mutant D128A thereof; 5) expressing uridine-5-monophosphate kinase or a mutant D93A thereof; and 6) expressing cytidine triphosphate synthetase or mutants D160E, E162A, E168K thereof.

10. The recombinant microorganism for producing citicoline according to claim 9, wherein the encoding gene of the transcription inhibition repressor protein of the encoding gene of the carbamoyl phosphate synthetase comprises one or a plurality of purR, pepA, and argR.

11. A method for producing citicoline by using a recombinant microorganism, wherein the citicoline is obtained by means of fermentation using the recombinant microorganism according to claim 1 with choline chloride added as a substrate.

12. The recombinant microorganism for producing citicoline according to claim 2, wherein the recombinant microorganism comprises Escherichia coli.

13. The recombinant microorganism for producing citicoline according to claim 3, wherein the recombinant microorganism comprises Escherichia coli.

14. The recombinant microorganism for producing citicoline according to claim 4, wherein the recombinant microorganism comprises Escherichia coli.

15. The recombinant microorganism for producing citicoline according to claim 5, wherein the recombinant microorganism comprises Escherichia coli.

16. A method for producing citicoline by using a recombinant microorganism, wherein the citicoline is obtained by means of fermentation using the recombinant microorganism according to claim 2 with choline chloride added as a substrate.

17. A method for producing citicoline by using a recombinant microorganism, wherein the citicoline is obtained by means of fermentation using the recombinant microorganism according to claim 3 with choline chloride added as a substrate.

18. A method for producing citicoline by using a recombinant microorganism, wherein the citicoline is obtained by means of fermentation using the recombinant microorganism according to claim 4 with choline chloride added as a substrate.

19. A method for producing citicoline by using a recombinant microorganism, wherein the citicoline is obtained by means of fermentation using the recombinant microorganism according to claim 5 with choline chloride added as a substrate.

20. A method for producing citicoline by using a recombinant microorganism, wherein the citicoline is obtained by means of fermentation using the recombinant microorganism according to claim 6 with choline chloride added as a substrate.

21. A method for producing citicoline by using a recombinant microorganism, wherein the citicoline is obtained by means of fermentation using the recombinant microorganism according to claim 7 with choline chloride added as a substrate.

22. A method for producing citicoline by using a recombinant microorganism, wherein the citicoline is obtained by means of fermentation using the recombinant microorganism according to claim 8 with choline chloride added as a substrate.

23. A method for producing citicoline by using a recombinant microorganism, wherein the citicoline is obtained by means of fermentation using the recombinant microorganism according to claim 9 with choline chloride added as a substrate.

24. A method for producing citicoline by using a recombinant microorganism, wherein the citicoline is obtained by means of fermentation using the recombinant microorganism according to claim 10 with choline chloride added as a substrate.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIG. 1 is a schematic diagram of a metabolic pathway of citicoline in Escherichia coli; and

[0031] FIG. 2 is an HPLC spectrogram of detection of the citicoline.

[0032] Carbamoyl phosphate: Carbamoyl phosphate; carbamoyl aspartate: Carbamoyl aspartate; orotate: Orotate; OMP: Orotate-5-monophosphate; UMP: Uridine-5-monophosphate; UDP: Uridine-5-diphosphate; UTP: Uridine-5-triphosphate; UdR: Uridine; CdR: Cytidine; CTP: Cytidine-5-triphosphate; CDP: Cytidine-5-Diphosphate; CMP: Cytidine-5-monophosphate; CDPC: Citicoline; PC: Phosphocholine; Choline: Choline; glycine betaine: Glycine betaine; PyrA: Carbamate phosphate synthetase; PyrB/I: Aspartate carbamyltransferase; PyrE: Orotate phosphoribosyltransferase; PyrF: Uridine-5 phosphate decarboxylase; PyrH: Uridine-5-monophosphate kinase; PyrG: Cytidine triphosphate synthetase; Cmk: Cytidine-5-monophosphate kinase; CTase: Cytidylyltransferase; CKase: Choline kinase; Ndk: Nucleoside diphosphate kinase; NudG: 5-hydroxy-cytidine triphosphate diphosphatase; UMP: Uridine-5-monophosphate; UmpG: non-specific nucleotide enzyme/polyphosphatase; UmpH: Uridine 5-phosphatase; BetA: Choline dehydrogenase; BetB: Betaine aldehyde dehydrogenase; AphA: Acid phosphatase; PhoA: alkaline phosphatase; UshA: UDP-sugar hydrolase; Cdh: Cytidine diphosphate-diacylglycerol pyrophosphatase.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] The present invention will be further illustrated below by means of several specific examples, which are only for the purpose of illustration instead of limitation.

Example 1: Method of Knocking Out a Gene in Escherichia coli

[0034] In the present invention, a Datsenko method was used to knock out a gene in Escherichia coli (Datsenko KA 2000, Proc Natl Acad Sci USA, 97(12):6640-6664), and for a corresponding gene knockout primer, reference is made to Baba T. 2006, Mol Syst Biol 2(1), 0008.

Example 2: Method for Verifying a Recombinant Strain Through Shake Flask Fermentation

[0035] In a fermentation medium for verifying production of citicoline by the recombinant strain through shake flask fermentation, each liter of the medium specifically includes 100 ml of a YC solution, 20 g of glucose, 200 ml of a 5-fold salt solution, 1 ml of a TM2 solution, 10 mg of ferric citrate, 120 mg of anhydrous magnesium sulfate, 111 mg of calcium chloride, and 1 ug of thiamine, with deionized water being used to bring the mixture to a required volume, wherein the 5-fold salt solution consists of 30 g of disodium hydrogen phosphate per liter, 15 g of potassium dihydrogen phosphate per liter, 2.5 g of sodium chloride per liter, and 5.0 g of ammonium chloride per liter, with ionized water being used to bring the mixture to a required volume; and the TM2 solution consists of 2.0 g of zinc chloride tetrahydrate per liter, 2.0 g of calcium chloride hexahydrate per liter, 2.0 g of sodium molybdate dehydrate per liter, 1.9 g of copper sulfate pentahydrate per liter, 0.5 g of boric acid per liter, and 100 ml of hydrochloric acid per liter. A YC solution in the fermentation medium M9 is 100 ml of deionized water; and a YC solution in the fermentation medium MS3.2 consists of 4 g of peptone, 4 g of yeast powder, 10 g of sodium chloride, and 100 ml of deionized water. The above solutions were sterilized at 121 C. for 20-30 minutes.

[0036] A shake flask fermentation process is as follows: first, the recombinant strain was inoculated into an LB medium of a certain amount and containing antibiotics (Molecular Cloning: A Laboratory Manual, written by [US] J. Sambrook, translated by Huang Peitang, 2002, 1595), the seeded medium was placed in a 34 C. shaker for overnight incubation at 250 rpm; 100 l of the above overnight seed culture was taken and transferred to 2 ml LB containing antibiotics, and then was placed in a 34 C. shaker for incubation of 4-6 h at 250 rpm, until an OD.sub.600 value is about 1.5; after that, the 2 ml of a secondary seed culture was entirely transferred into a shake flask pre-loaded with 18 ml of the fermentation medium, and placed in a 34 C. shaker for incubation at 250 rpm. When OD.sub.600 value of the culture reached to 1, IPTG was added for a final concentration of 0.1 mM, then choline chloride was added for a final concentration of 4 mM, the incubation continues for about 20 hours, and a fermentation broth was taken for centrifugation detection, wherein for a specific detection method, reference is made to Example 4.

Example 3: Method for Producing Citicoline Through Fermentation Using a Recombinant Strain in a 5 L Fermenter

[0037] In a fermentation medium for verifying production of citicoline by the recombinant strain through fermentation in a fermenter, each liter of the medium specifically includes 2 g of ammonium sulfate, 8 g of sodium chloride, 2 g of potassium dihydrogen phosphate, 1.65 g of magnesium chloride hexahydrate, 10 g of glucose, 105 mg of calcium chloride, 10 mg of zinc chloride, 1 mL of a TM2 trace element solution, 94 mg of iron citrate, 6 g of peptone, and 6 g of yeast powder, with deionized water being used to bring the mixture to a required volume. The TM2 trace element solution consists of 1.31 g of zinc chloride per liter, 1.01 g of calcium chloride per liter, 1.46 g of ammonium molybdate tetrahydrate per liter, 1.9 g of copper sulfate per liter, 0.5 g of boric acid per liter, and 10 mL of hydrochloric acid per liter, with deionized water being used to bring the mixture to a required volume. A supplementary medium contains 600 g of glucose, 40 g of peptone, and 40 g of yeast powder per liter.

[0038] A fermentation process is as follows: first, a seed culture was prepared, a monoclonal culture was picked up from an LB plate and transferred to an LB tube containing antibiotics for overnight incubation at 34 C., the obtained culture was incubated, at an inoculation volume of 1%, into a 500 mL shake flask loaded with 100 mL LB for incubation at 34 C. for 4 hours, until an OD value was 1.5-2; then, the obtained culture was incubated, at an inoculation volume of 5%, into a 5 L fermenter loaded with 1.5 L of fermentation medium MF1.32 for incubation at 37 C., a pH value was adjusted to 6.9 with ammonia water, oxygen dissolution was coupled with a rotation speed to maintain dissolved oxygen at 30%, after 3 hours of fermentation, additional medium was fed at a rate of 8 g/L/h, after 5 hours of fermentation, an OD600 value was 16-25, the temperature was reduced to 32 C., IPTG was added such that a final concentration reaches 0.5 mmol/L for induction, after 10 hours of fermentation, the rotation speed was fixed to 1000 rpm, and when the dissolved oxygen was higher than 40%, coupling feed started, so as to maintain the dissolved oxygen at 30%-45%. Sampling and detection were performed after 27 hours of fermentation, and for a detection method, reference is made to Example 4.

Example 4: Measurement of Citicoline and Related by-Products in a Fermentation Broth Through HPLC

[0039] 200 ul of a fermentation broth was precisely extracted, and added into 800 ul of deionized water, to which 1 ml of absolute ethanol is added, the obtained mixture was subject to vortex shaking for 5 min (at 10,000 rpm), after centrifugation, a supernatant was taken and filtered by a 0.22 um filter membrane, and high-performance liquid chromatography (HPLC) was used for detection, with the following HPLC parameters: Agilent SB C18 4.6*150 mm 5 um is adopted; mobile phases are methanol and 10 mM PBS (pH4.0); the ratio of the mobile phases is as follows: in 0.01-4.00 minutes, the proportion of the methanol is 2%, in 4.00-5.00 minutes, the proportion of the methanol is raised from 2% to 10%, in 4.00-5.00 minutes, the proportion of the methanol is reduced from 10% to 2%, and in 5.10-10.0 minutes, the proportion of the methanol is 2%; a wavelength of 272 nm is detected by an ultraviolet detector; the flow rate of the mobile phase is 0.8 mL/min, the loading amount of the fermentation broth is 5 L, and the column temperature is 30 C. The CDPC peak time is 2.745 minutes, and the spectrogram is as shown in FIG. 2.

Example 5: Construction of Recombinant Escherichia coli Incapable of Degrading and Utilizing CDPC

[0040] As shown in FIG. 1, in Escherichia coli, CDPC is catalyzed by UDP-sugar hydrolase (UshA) and CDP-diacylglycerol pyrophosphatase (Cdh) to generate CMP and phosphocholine. In the present invention, Escherichia coli WJ2 (ATCC27325lacI entDT5) was used as an original strain, and encoding genes of UshA and Cdh were knocked out from WJ2 to obtain recombinant bacteria. The ability of these strains to utilize and degrade CDPC was gradually weakened, wherein a recombinant strain HQ24 (WJ2ushAumpGumpHpyrIcdh) cannot utilize and degrade CDPC in shake flask fermentation. For example, when 2.0 g/L citicoline dicholine is added to the fermentation medium, CDPC residues in WJ2 and WJ3 (WJ2ushA) detected after 24 h are 0.15 g/L and 1.02 g/L. [SQ150916 result: 0.3/1.92] indicates that knocking out ushA significantly reduced degradation of CDPC. When 1 g/L citicoline diphosphate is added to the fermentation medium, CDPC residues in HQ19 (WJ2ushAumpGumpH pyrI), HQ24 (HQ19cdh), HQ25 (HQ19mazG), and HQ26 (HQ19nudF) detected after 24 h are 0.5 g/L, 1.0 g/L, 0.45 g/L, and 0.46 g/L. It indicates that knocking out cdh could completely eliminate the degradation of CDPC. HQ24/pY022 (pHS01-PCT1-CKI1-pyrE-prs.sup.128) reached around 17 g/L after fermentation in a fermenter for 26 h.

[0041] The recombinant strain HQ24/pY022 strain was deposited on Jun. 26, 2017 at China General Microbiological Culture Collection Center of China Microbe Preservation Management Committee, Address: NO. 3, NO. 1 Courtyard West Beichen Road, Chaoyang District, Institute of Microbiology, Chinese Academy of Sciences, Postcode: 100101, Preservation number: CGMCC No. 14277.

Example 6: Construction of Recombinant Escherichia coli Incapable of Degrading Choline

[0042] In the present invention, for metabolic production of citicoline by Escherichia coli, exogenously added choline chloride was used as a substrate as the precursor for citicoline synthesis. Under the action of choline dehydrogenase, the substrate choline is oxidized to betaine aldehyde, and then further to glycine betaine under the action of betaine aldehyde dehydrogenase.

[0043] In order to prevent degradation of the substrate and the intermediate product, in the present invention, genes betA/B encoding choline dehydrogenase were both knocked out in HQ34 strain.

[0044] HQ33 (HQ24purRfhuA::Ptrc-pyrE, carB948) and HQ34 (HQ33betAB) were respectively used as hosts to express the plasmid pY012. After fermentation in a fermenter for 43 h, the detected yields of CDPC were 18.40 g/L for HQ33/pY012 and 20.87 g/L for HQ34/pY012. It indicates that knocking out choline dehydrogenase could block degradation of the substrate choline chloride, thereby increased the yield of CDPC.

Example 7: Construction of Recombinant Escherichia coli Incapable of Degrading Phosphocholine

[0045] In the present invention, for production of citicoline, exogenously added choline chloride was used as a substrate, which is catalyzed by choline kinase to generate an intermediate product, i.e., phosphocholine. However, phosphocholine in Escherichia coli is prone to hydrolysis to choline by acid phosphatase or alkaline phosphatase present in a cell.

[0046] In order to prevent degradation of the intermediate product phosphocholine, in the present invention, coding genes of AphA phoA of were knocked out in HQ35 and HQ36, respectively.

[0047] HQ33 (HQ24purRfhuA::Ptrc-pyrE, carB948), HQ35 (HQ33aphA), and HQ36 (HQ33phoA) were used as hosts to express the plasmid pY012. After shake flask fermentation in a fermentation medium, the detected yields of CDPC in above strains were 1.82 g/L, 1.86 g/L, and 1.87 g/L, respectively. It indicates that knocking out phosphatase could reduce or block the degradation of the intermediate product phosphocholine, such that more phosphocholine could be used for product synthesis, thereby increasing the yield of CDPC.

Example 8: Overexpression of Cytidylyltransferase and/or Choline Kinase for Increasing the Yield of CDPC

[0048] When degradation genes of citicoline and of the substrate choline and the intermediate product phosphocholine thereof are knocked out, the yield of CDPC is very low, indicating insufficient expression of a key intracellular enzyme for catalyzing choline to generate CDPC. In order to increase the yield of CDPC in a cell, in the present invention, CTase and CKase in the CDPC synthesis process were expressed appropriately. When plasmids expressing different genes encoding CTase and/or CKase were transformed into Escherichia coli expression hosts, the yield of CDPC was increased in varying degrees. For example, when plasmids pHS01 (pCL1920 PLac:: Ptrc, see LS9 patent US20130029395A1), pY008 (pHS01-PCT1-CKI1), and pY012 (pHS01-PCT1-CKI1-licC) are expressed in HQ33, after shake flask fermentation culture for 24 hours, the yields of CDPC are respectively 0 g/L, 1.44 g/L, and 1.55 g/L.

Example 9: Overexpression of Choline Transporter Protein for Increasing the Yield of CDPC

[0049] BetT is a choline transporter protein driven by hydrogen ions and belongs to the family of betaine choline transporter BCCT. Said protein is expressed in an aerobic condition and induced by osmotic pressure. High osmotic pressure can enhance transcription of BetT, and addition of choline can further enhance the transcription. In order to obtain more CDPC, choline chloride needs to be supplied more quickly. In order to increase entering of the choline chloride into a cell, which is a rate-limiting step, BetT was overexpressed, so as to obtain more transporter protein in a case where the osmotic pressure was not increased. HQ33 was used as a host to respectively express pY012 and pY012-betT, and after shake flask fermentation in a fermentation medium for 24 h, the detected yield of CDPC in the former case was 2.36 times of that in the latter case. It indicates that overexpression of betT could significantly increase the yield of CDPC.

Example 10: Knocking Out of an Encoding Gene of UMP Degrading Enzyme for Increasing the Yield of CDPC

[0050] Uridine monophosphate UMP is an intermediate product of the pyrimidine synthesis pathway, can generate uridine diphosphate UDP under the action of uridine 5-monophosphatase through a replenishment pathway, and can further be degraded into uridine under the action of 5-nucleotidase. In order to obtain a larger amount of the CDPC product, in the present invention, encoding genes umpG and umpH of the 5-nucleotidase were knocked out in HQ18. WJ3 and HQ18 (WJ3umpGumpH) were used as hosts to transform the plasmid pY022 (pY008-pyrE-prs.sup.128), and after shake flask fermentation in a fermentation medium for 24 h, the detected yields of CDPC were 1.9 g/L and 1.92 g/L, respectively. For a strain HQ18/pY022, a yield could reach 8.93 g/L after fermentation in a 5 liter fermenter for 26 h.

Example 11: Overexpression of PyrE for Increasing the Yield of CDPC

[0051] Overexpression of the pyrE gene in a plasmid and a genome could remove defect caused by a frameshift mutation of the upstream rph gene, thereby increase the yield of CDPC. For example, when HQ04 (W3110 (deoA ung purR ushA betABI, Ptrc-betT)) is used as a host to express pY008 and pY009 (pY008-pyrE), respectively, and after shake flask fermentation in a fermentation medium, the yield of CDPC is increased from 0.67 g/L to 0.80 g/L. It indicates that the overexpression of pyrE could increase the yield of citicoline.

Example 12: Overexpression of Prs128 for Increasing the Yield of CDPC

[0052] In the pyrimidine synthesis pathway of Escherichia coli, under the action of orotate phosphoribosyltransferase (PyrE), phosphate groups in phosphoribosylpyrophosphate (PRPP) are transferred to orotate, thereby generating orotate monophosphate (OMP). PRPP insufficiency may form a rate-limiting step in the pyrimidine synthesis pathway, resulting in a relatively low yield of citicoline and accumulation of an orotic acid by-product. Phosphoribosylpyrophosphate kinase (Prs) catalyzes a reaction between 5-phosphate ribose and ATP to synthesize PRPP. However, Prs is subject to feedback inhibition imposed by ADP and others, and a mutation from aspartic acid to alanine at position 128 in Prs for obtaining Prs128 could remove the feedback inhibition. For example, when WJ3 is used as a host to respectively express pY017 (pY009-prs) and pY022 (pY009-prs.sup.128), and after shake flask fermentation in a fermentation medium, the yield of CDPC is increased from 1.80 g/L to 1.96 g/L.

Example 13: Knocking Out of an Encoding Gene of PyrI for Increasing the Yield of CDPC

[0053] A second step of catalysis in the pyrimidine nucleotide synthesis pathway is catalyzing synthesis of carbamoyl aspartate by aspartate carbamyltransferase. Said enzyme consists of a regulatory subunit (PyrI) and a catalytic subunit (PyrB). When the concentration of CTP is high, CTP is combined with PyrI, thereby reducing the activity of the enzyme. In the present invention, the encoding gene of PyrI was knocked out, so as to remove the feedback inhibition effect of the end product CTP on the enzyme. For example, when pY022 is expressed in HQ18 and HQ19 (HQ18pyrI), which are used as hosts, and after shake flask fermentation in a fermentation medium, the yield of CDPC is increased from 1.74 g/L in HQ18 to 1.92 g/L in HQ19. It indicates that knocking out the gene of pyrI could remove the feedback inhibition on aspartate carbamyltransferase imposed by CTP, thereby accelerating synthesis of the pyrimidine synthesis pathway and increasing the yield of CDPC.

Example 14: Knocking Out of Encoding Genes of Repressor Proteins PurR and ArgR for Increasing the Yield of CDPC

[0054] A first step of the pyrimidine nucleotide synthesis pathway is catalyzing synthesis of carbamoyl phosphate by carbamoyl phosphate synthetase. The encoding gene carAB of said enzyme is subject to a feedback repression effect of metabolic end products such as purine, pyrimidine, and arginine. In addition, purine nucleotide is inhibited by repressor protein encoded by the purR gene, and arginine is inhibited by repressor protein encoded by the argR gene. In the present invention, transcription inhibition to carAB was removed by knocking out purR and argR, thereby accelerating synthesis of carbamoyl phosphate. For example, when pY022 is respectively expressed in HQ19,HQ22 (HQ19purR), HQ20 (HQ18purR) and HQ21 (HQ20argR), and after shake flask fermentation in the fermentation medium MS3.2, the yields of CDPC are 1.25 g/L and 1.54 g/L; and 1.15 g/L and 1.34 g/L, respectively. It indicates that knocking out purR or argR gene could remove the feedback repression to carAB, thereby promoting the synthesis of the pyrimidine pathway and increasing the yield of CDPC.

[0055] Only some of the embodiments of the present invention are described above. It should be noted that those skilled in the art could make some improvements without departing from the principle of the present invention, and these improvements shall also fall into the protection scope of the present invention.