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Recombinant microorganism for producing uridine and method for producing uridine

A technology for recombining microorganisms and uridine, applied in the biological field, can solve problems such as low yield and unstable fermentation, and achieve high promotion and application value

Pending Publication Date: 2021-12-07
SUZHOU BIOSYNTHETICA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

From the 1980s to the mid-1990s, Japanese scientists began to publish the results of the production of uridine by fermentation. Takeda Pharmaceutical Co., Ltd. used the bacillus obtained by mutagenesis to ferment uridine, but the yield was as low as 5g / L, and the fermentation was unstable. etc. (CN85107905)
Domestic research has not yet seen the report of scale-up production

Method used

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  • Recombinant microorganism for producing uridine and method for producing uridine

Examples

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Effect test

Embodiment 1

[0032] Example 1 The method for gene knockout in Escherichia coli

[0033] 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), corresponding gene knockout primers see Baba T2006Mol Syst Biol 2(1), 0008.

Embodiment 2

[0034] Embodiment 2 shaking flask fermentation verifies the method for recombinant bacterial strain

[0035] The fermentation medium for verifying that the recombinant strain produces uridine in the shake flask fermentation is 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 in each liter of medium 120mg, calcium chloride 111mg, thiamine 1ug, dilute to volume with deionized water. Among them, the 5 times salt solution is 30g per liter of disodium hydrogen phosphate, 15g per liter of potassium dihydrogen phosphate, 2.5g per liter of sodium chloride, 5.0g of ammonium chloride, constant volume with ion water; TM2 solution is tetrahydrate chloride Zinc 2.0g per liter, calcium chloride hexahydrate 2.0g per liter, sodium molybdate dihydrate 2.0g per liter, copper sulfate pentahydrate 1.9g per liter, boric acid 0.5g per liter, hydrochloric acid 100ml per liter. YC solution...

Embodiment 3

[0037] Embodiment 3 5L fermentor utilizes the method for fermenting and producing uridine with recombinant bacterial strains

[0038] Verify that the fermentation medium for recombinant strains to ferment and produce uridine 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 105mg, zinc chloride 10mg, TM2 trace element solution 1mL, ferric citrate 9.4mg, peptone 0-10g, yeast powder 0-10g, dilute to volume with deionized water. Among them, the TM2 trace element solution is 1.31g of zinc chloride, 1.01g of calcium chloride, 1.46g of ammonium molybdate tetrahydrate, 1.9g of copper sulfate, 0.5g of boric acid, and 10mL of hydrochloric acid, and the volume is constant with deionized water. 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 uridine and a method for producing uridine by using the recombinant microorganism. Wherein the uridine is degraded and knocked out by using genes, and the genes encode ribonucleoside hydrolase, uridine phosphorylase, nucleoside phosphorylase, cytidine / uridine kinase and nucleoside transporter protein. Meanwhile, overexpression is carried out on key enzymes in a uridine biosynthetic pathway, including cytidine triphosphate pyrophosphorylase for degrading cytidine triphosphate to cytidine monophosphate and uridine monophosphate phosphorylase for catalyzing uridine monophosphate to uridine. In addition, a pyrimidine nucleoside pathway is subjected to genetic engineering modification, and feedback inhibition of a synthetic pathway is relieved. The recombinant strain can reach the uridine yield of 20 g / L or above in a 5-liter fermentation tank through a biological fermentation method, industrial mass production can be achieved, meanwhile, the uridine production cost is low, pollution is reduced, the method is green and environmentally friendly, and the method has high application and popularization value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a recombinant microorganism for producing uridine, using the recombinant microorganism to produce uridine and using the recombinant microorganism to produce uridine through a biological method. Background technique [0002] Uridine, also known as uridine nucleoside, is a kind of nucleoside, which is white needle-like crystal or powder, odorless, slightly sweet and slightly pungent. Soluble in water, slightly soluble in dilute alcohol, insoluble in anhydrous alcohol. [0003] Molecular formula C 9 h 12 N 2 o 6 , The relative molecular mass is 224.2, the melting point is 163-167°C, and the specific rotation is 8.4°. It is mainly used for giant red blood cell anemia, and it can also be used in combination with other nucleosides and bases to treat liver, cerebrovascular and cardiovascular diseases. It is also used to manufacture fluorouracil (S-FC), deoxynucleosides, and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P19/38C12R1/19C12R1/125C12R1/15C12R1/225
CPCC12N9/1241C12N9/1077C12N9/2497C12Y204/02003C12Y302/02003C12P19/385
Inventor 江君君田锋王欣彤胡志浩
Owner SUZHOU BIOSYNTHETICA CO LTD
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