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A recombinant microorganism for producing citicoline and a method for producing citicoline

A technology for the production of citicoline and recombinant microorganisms, applied in the biological field, can solve the problems of unfavorable industrial production application, high cost, low conversion rate, etc., and achieve the effect of realizing large-scale industrial production and reducing production costs

Active Publication Date: 2019-01-15
SUZHOU BIOSYNTHETICA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, using yeast or Brevibacterium ammoniagenes as substrates to produce citicoline with orotic acid and phosphorylcholine as substrates, adding glucose to produce ATP, still has high cost and low conversion rate. The problem (Ying Hanjie 2015, A strain of genetically engineered bacteria expressing choline kinase and phosphorylcholine cytidine transferase and its construction method and application, 201510184705.8) is not conducive to large-scale industrial production and application

Method used

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  • A recombinant microorganism for producing citicoline and a method for producing citicoline
  • A recombinant microorganism for producing citicoline and a method for producing citicoline

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Embodiment 1

[0035] Example 1 The method for gene knockout in Escherichia coli

[0036] The present invention uses Datsenko's method to knock out genes in Escherichia coli (Datsenko KA 2000, ProcNatl Acad Sci USA, 97(12): 6640-6645), see Baba T2006, Mol SystBiol 2(1) for the corresponding gene knockout primers ,0008.

Embodiment 2

[0037] Example 2 Shake flask fermentation method for verifying recombinant strains

[0038] The fermentation medium for verifying that the recombinant strain produces citicoline in shake flask fermentation is specifically 100ml of YC solution per liter of medium, 20g of glucose, 200ml of 5 times salt solution, 1ml of TM2 solution, 10mg of ferric citrate, and no Magnesium sulfate water 120mg, calcium chloride 111mg, thiamine 1ug, constant volume with deionized water, 5 times of the salt solution is disodium hydrogen phosphate 30g per liter, potassium dihydrogen phosphate 15g per liter, sodium chloride per liter 2.5g, ammonium chloride 5.0g, constant volume with ion water; TM2 solution is zinc chloride tetrahydrate 2.0g per liter, calcium chloride hexahydrate 2.0g per liter, sodium molybdate dihydrate 2.0g per liter, pentahydrate Copper sulfate is 1.9g per liter, boric acid is 0.5g per liter, and hydrochloric acid is 100ml per liter. The YC solution in the fermentation medium M...

Embodiment 3

[0040] Example 3 5L fermenter using recombinant strains to ferment and produce citicoline

[0041] Verify that the fermentation medium for recombinant strains to ferment and produce citicoline in a fermenter is MF1.32, specifically containing 2 g of ammonium sulfate, 8 g of sodium chloride, 2 g of potassium dihydrogen phosphate, and 1.65 g of magnesium chloride hexahydrate in each liter of medium. g, glucose 10g, calcium chloride 105mg, zinc chloride 10mg, TM2 trace element solution 1mL, ferric citrate 94 mg, peptone 6g, yeast powder 6g, dilute to volume with deionized water. Among them, the TM2 trace element solution is zinc chloride 1.31 g, calcium chloride 1.01 g, ammonium molybdate tetrahydrate 1.46 g, copper sulfate 1.9 g, boric acid 0.5 g, hydrochloric acid 10 mL, and deionized water to volume. The feeding medium contains 600g of glucose, 40g of peptone and 40g of yeast powder per liter.

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Abstract

The invention provides a recombinant microorganism for producing citicoline and a method for producing citicoline by using the recombinant microorganism, which knocks out the degradation and utilization genes of citicoline, choline and phosphorylcholine, and knocks out the 5-hydroxyl-CTP bisphosphatase encoded by the CTP degradation gene, thus blocking the shunt of CTP, the precursor of CDP. The key enzymes involved in citicoline synthesis are expressed in appropriate amounts, including choline kinase which phosphorylates choline chloride to phosphorylcholine, and choline phosphocytidyltransferase, which catalyzes the reaction of phosphorylcholine with CDP to produce citicoline. In addition, the pyrimidine nucleoside pathway was genetically engineered to remove the feedback inhibition of the synthesis pathway. The recombinant strain could produce more than 20g / L citicoline in a 5-liter fermentor by biological fermentation, and the production cost of citicoline is low, the pollution islittle and the environment is green, so it had high value of popularization and application.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a recombinant microorganism for producing citicoline, using the recombinant microorganism to produce citicoline and using the recombinant microorganism to produce citicoline through a biological method. Background technique [0002] Citicoline (CDP-choline, abbreviated as CDPC) is a nucleoside derivative, which is synthesized from 5' cytidylic acid and phosphorylcholine. Molecular formula C 14 h 26 N 4 o 11 P 2 , relative molecular mass 488.323962, boiling point 851.4°C. It is an intermediate of phosphatidylcholine from choline, exists in all cells, and is the main coenzyme of phospholipid synthesis, improving brain function by promoting the synthesis of lecithin. For the treatment of acute traumatic brain injury and disturbance of consciousness after brain surgery. [0003] The production of citicoline mainly has two kinds of chemical method and biological method....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P19/30C12R1/19
CPCC12N9/1229C12N9/93C12P19/30C12Y207/04022C12Y603/04016C12N9/16C12N9/14C12N9/0006C12N9/1205C12N9/1241C07K14/245C12N15/52C12P19/305C12Y301/03005C12Y306/01026C12Y101/99001C12Y301/03001C12Y301/03002C12Y207/01032C12Y207/07015C12N1/20C12N15/70
Inventor 胡志浩江君君范俊英刘爱霞秦天宇田锋张凯林王欣彤
Owner SUZHOU BIOSYNTHETICA CO LTD
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