Recombinant microorganism for producing cytidine and method for producing cytidine

A technology of recombinant microorganism and cytidine, applied in the field of cytidine production, can solve the problems of unknown strain genotype, low cytidine yield, poor repeatability, etc., and achieve the effect of high popularization and application value.

Active Publication Date: 2017-05-31
BIOSYNTHETICA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The cytidine yields of these strains are too low to meet the requirements of industrial production. Although Asahi's mutagenesis screening strains

Method used

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  • Recombinant microorganism for producing cytidine and method for producing cytidine
  • Recombinant microorganism for producing cytidine and method for producing cytidine

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Example 1 Method for gene knockout in Escherichia coli.

[0032] The present invention adopts Datsenko's method to carry out gene knockout in Escherichia coli (Datsenko KA2000.Proc Natl Acad Sci USA, 97(12):6640-6645), and the corresponding gene knockout primers are shown in BabaT2006 Mol Syst Biol 2(1), 0008.

Embodiment 2

[0033] Example 2 Shake Flask Fermentation A method for verifying recombinant strains.

[0034] Fermentation medium for verifying the production of cytidine by recombinant strains in shake flask fermentation, specifically 100ml of YC solution, 20g of glycerol, 200ml of 5 times salt solution, 1ml of TM2 solution, 10mg of ferric citrate, and anhydrous magnesium sulfate per liter of medium 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 2.5g per liter, Ammonium chloride 5.0g, constant volume with ion water; TM2 solution is 2.0g per liter of zinc chloride tetrahydrate, 2.0g per liter of calcium chloride hexahydrate, 2.0g per liter of sodium molybdate dihydrate, and 2.0g per liter of copper sulfate pentahydrate. Lit 1.9g, boric acid 0.5g per liter, hydrochloric acid 100ml per liter. The YC solution in the ferment...

Embodiment 3

[0036] Example 3 A method for fermenting and producing cytidine using a recombinant strain in a 5L fermenter.

[0037]Verify that the fermentation medium for recombinant strains to ferment and produce cytidine in a fermenter is MF1.16, specifically, each liter of medium contains 10g of ammonium sulfate, 2g of sodium chloride, 2g of potassium dihydrogen phosphate, 2g of magnesium sulfate heptahydrate, and glycerol 25g, calcium chloride 105 mg, zinc chloride 10mg, TM2 trace element solution 1mL, ferric citrate 9.4 mg, peptone 0-10g, yeast powder 0-10g, dilute to volume with deionized water. The TM2 trace element solution is 1.31 g of zinc chloride, 1.01 g of calcium chloride, 1.46 g of ammonium molybdate tetrahydrate, 1.9 g of copper sulfate, 0.5 g of boric acid, and 10 mL of hydrochloric acid. The feeding medium contains 500g of glycerol, 0-10g of peptone and 0-10g of yeast powder per liter.

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Abstract

The invention provides a recombinant microorganism for producing cytidine and a method for producing the cytidine from the recombinant microorganism. A degradation and use gene of the cytidine is knocked off, and the gene encodes cytidine ammonialyase, ribonucleotide hydrolase, cytidine/uridine kinase and nucleoside transporter. Meanwhile key enzymes in biological synthesis process of the cytidine are over-expressed, including degrading cytidine triphosphoric acid to cytidine triphosphoric acid pyrophosphorylase of the cytidine monohosphoric acid, and catalyzing the cytidine monohosphoric acid to cytidine monohosphoric acid phosphorylase of the cytidine. In addition, pyrimidine nucleoside process is subjected to genetic engineering reform, and feedback inhibition of the synthesis process is relieved. By using a biological fermentation method, the cytidine yield greater than 20g/L can be achieved for a recombinant strain in a fermentation tank of 5L, industrial on-scale production can be achieved, and meanwhile the recombinant microorganism is low in cytidine production cost, small in pollution, green and environmental-friendly and relatively high in popularization and application value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a recombinant microorganism producing cytidine, producing cytidine by using the recombinant microorganism and producing cytidine by using the recombinant microorganism through a biological method. Background technique [0002] Cytidine is a kind of nucleoside, which is synthesized from cytosine and ribose, and the two are connected by β-N1-glycosidic bonds. White or off-white crystal or powder; soluble in water, soluble in acid and alkali, slightly soluble in ethanol, insoluble in organic solvents. Molecular formula C 9 h 13 N 3 o 5 , The relative molecular mass is 243.22, the melting point is 210.0~220.0, and the specific rotation is +31~+35°. It is mainly used in the preparation of cytidine triphosphate (CTP), citicholine (sodium salt) (Citicholine, CDP-choline), 2′, 3′-dideoxycytidine and cytarabine and other raw materials. [0003] At present, the production of ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P19/38C12R1/19C12R1/125C12R1/15C12R1/225
CPCC12N9/1205C12N9/2497C12N9/78C12P19/38C12Y207/01048C12Y302/02003C12Y305/04005
Inventor 刘爱霞胡志浩范俊英江君君田锋王欣彤
Owner BIOSYNTHETICA INC
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