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Recombinant microorganism for producing cytosine and method for producing cytosine

A technology for recombining microorganisms and cytosine, which is applied in the biological field and can solve problems such as the lack of industrial production by microbial methods

Active Publication Date: 2021-02-12
SUZHOU BIOSYNTHETICA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] 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 microorganism for producing cytosine and method for producing cytosine
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  • Recombinant microorganism for producing cytosine and method for producing cytosine

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Effect test

Embodiment 1

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

[0036] The present invention adopts Datsenko's method to carry out gene knockout in Escherichia coli (Datsenko 2000), and see Baba 2006 for the corresponding gene knockout primers.

Embodiment 2

[0037] Embodiment 2 shaking flask fermentation verifies the method for recombinant bacterial strain

[0038] 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

[0040] Embodiment 3HPLC measures cytidine and cytosine in fermented liquid

[0041] 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 SBC18 4.6*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%, 3.50-3.60 minutes The proportion of methanol is reduced from 10% to 2%, and the proportion of methanol is 2% in 3.60-8.5 minutes. It is detected by an ultraviolet detector and the detection wavelength is 260nm; the flow rate of the initial mobile phase is 1.0mL / min, and the sample volume of the fermentation broth is 5 μL. The column temperature is 30°C.

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Abstract

The invention provides a recombinant microorganism for producing cytosine. The recombinant microorganism is classified and named as Escherichia coli., the strain is preserved in China General Microbiological Culture Collection Center on May 08, 2019, the address is Institute of Microbiology, Chinese Academy of Sciences, No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the postcodeis 100101; and the preservation number of the strain is CGMCC No. 17730. The recombinant microorganism for producing the cytosine is fermented to obtain the cytosine; or nucleoside hydrolase is addedinto the cytidine fermentation liquor or the solution containing cytidine to produce cytosine.

Description

[0001] This application is a divisional application of the invention patent application with the application number: 201910602231.2, the invention name: recombinant microorganisms producing cytosine and the method for producing cytosine, and the application date: July 05, 2019. technical field [0002] 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 [0003] 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 ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
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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