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Recombinant microbe for producing cytosine and method for producing cytosine

A technology for recombining microorganisms and cytosine, applied in the biological field, can solve the problem of not realizing the industrial production of microorganisms

Inactive Publication Date: 2019-11-05
SUZHOU BIOSYNTHETICA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Humans have used microorganisms to ferment and produce nucleoside products, such as β-thymidine, guanosine, inosine and adenosine, but cytidine and cytosine have not been industrially produced by microbial methods

Method used

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  • Recombinant microbe for producing cytosine and method for producing cytosine
  • Recombinant microbe for producing cytosine and method for producing cytosine
  • Recombinant microbe for producing cytosine and method for producing cytosine

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Example 1 The method for gene knockout in Escherichia coli

[0035] The present invention uses Datsenko's method to knock out genes in Escherichia coli (Datsenko 2000), see Baba 2006 for the corresponding gene knockout primers.

Embodiment 2

[0036] Embodiment 2 shaking flask fermentation verifies the method for recombinant bacterial strain

[0037] Verification of the fermentation medium for recombinant strains to produce cytosine in shake flask fermentation, specifically 100ml of YC solution per liter of medium, 200ml of 5 times salt solution, 1ml of TM2 solution, 10mg of ferric citrate, 120mg of anhydrous magnesium sulfate, chlorine Calcium 111mg, thiamine 1ug, constant volume with deionized water, 5 times of the salt solution contains 30g of disodium hydrogen phosphate, 15g of potassium dihydrogen phosphate, 2.5g of sodium chloride, 5.0g of ammonium chloride per liter , constant volume with ion water; each liter of TM2 solution contains 2.0g of zinc chloride tetrahydrate, 2.0g of calcium chloride hexahydrate, 2.0g of sodium molybdate dihydrate, 1.9g of copper sulfate pentahydrate, 0.5g of boric acid, 100ml of hydrochloric acid . The YC solution in the fermentation medium is 2g of glycerol, 0.6g of peptone, 0.2...

Embodiment 3

[0039] Example 3 HPLC Determination of Cytidine and Cytosine in Fermented Broth

[0040] Draw 1ml of fermentation broth into a 2ml centrifuge tube, heat at 100°C for 5 minutes, cool to room temperature, dilute to a certain multiple, centrifuge through a 0.22μm filter membrane, and detect with high performance liquid chromatography (HPLC). The parameters of HPLC are as follows: Agilent SB C184.6*150mm 5μm, the mobile phase is methanol and 10mM PBS (pH4.0), the proportion of mobile phase is 0.01-2.80 minutes, the proportion of methanol is 2%, 2.80-3.50 minutes, the proportion of methanol is increased from 2% to 10%, 3.50- 3. The proportion of methanol was reduced from 10% to 2% in 60 minutes, and the proportion of methanol was 2% in 3.60-8.5 minutes. It was detected by an ultraviolet detector, and the detection wavelength was 260nm; the flow rate of the initial mobile phase was 1.0mL / min, and the loading amount of the fermentation broth was 5 μL, the column temperature is 30°C. ...

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Abstract

The invention discloses a recombinant microbe for producing cytosine and a method for producing the cytosine. The recombinant microbe has at least one of the following characteristics: (1) the recombinant microbe does not degrade or utilize the cytosine; (2) the recombinant microbe accumulates the cytosine externally; (3) the recombinant microbe overexpresses the gene of encoding ribose kinase; (4) the recombinant microbe can utilize glucose and ribose simultaneously; and (5) the gene of regulatorprotein of encoding ribose and related compounds of the recombinant microbe is deleted or has no function. The recombinant microbe culture is adopted, and the cytosine or a mixture of the cytosine and cytidine can be solely produced through fermentation

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for producing cytosine by a biological fermentation method and an enzymatic method. It is mainly the construction of recombinant microorganisms producing cytosine and the establishment of fermentation production methods, followed by the method of using genetically engineered microorganisms to produce cytidine hydrolase enzyme liquid, and then using the enzyme liquid to hydrolyze cytidine to produce cytosine. Background technique [0002] Cytosine is one of the pyrimidine bases in nucleic acids, scientific name is 4-amino-2-carbonylpyrimidine, CAS RN: 71-30-7, molecular formula: C 4 h 5 N 3 O. White or off-white crystalline powder, low solubility in water at room temperature, slightly soluble in ethanol, insoluble in ether. [0003] Cytosine is an important intermediate in fine chemicals, pesticides and medicine, especially in the field of medicine, mainly use...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P17/12C12R1/19
CPCC12N9/78C12Y305/04001C07K14/245C12N9/2497C12Y302/02008C12N9/1077C12Y204/02002C12N9/1205C12Y207/01015C12P17/12
Inventor 张凯林胡志浩江君君田锋王欣彤
Owner SUZHOU BIOSYNTHETICA CO LTD
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