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S. cerevisiae strain for producing citicoline through bioconversion and application of S. cerevisiae strain

A technology of Saccharomyces cerevisiae strains and microbial strains, which is applied in the field of biomedicine and can solve problems such as complex processes, low conversion rates, and unstable conversion rates

Active Publication Date: 2013-12-11
NANTONG QIUZHIYOU BIOSCI & BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since CMP has been mass-produced in a variety of ways, the price has dropped greatly, and there is no advantage in my country's double-bacteria fermentation compared with yeast biotransformation
Moreover, the dual-bacteria fermentation still has bottlenecks that are difficult to overcome, such as complex process, low conversion rate, and high separation difficulty.
The use of waste beer yeast biotransformation has defects such as difficulty in collecting yeast, large fluctuations in catalytic activity of waste yeast, and unstable conversion rate.

Method used

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  • S. cerevisiae strain for producing citicoline through bioconversion and application of S. cerevisiae strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Screening of Saccharomyces cerevisiae strains with high activity CMP kinase and high activity phosphorylcholine cytidylyltransferase

[0027] 1. Strains

[0028] Saccharomyces cerevisiae (S.cerevisiae QY131, strain preservation number CGMCC7978) was obtained from CGMCC2842 after mutagenesis.

[0029] 2. Medium

[0030] (1) Original medium A: 3% glucose, 0.3% beef extract, 0.3% yeast extract, 0.5% urea, FeSO 4 ·7H 2 O0.01%, agar 2.5%.

[0031] (2) Medium B for KCl sensitive strain screening: add 1.5mol / L KCl to medium A.

[0032] 3. Strain mutagenesis screening method

[0033] Dissolve diethyl sulfate (DES) stock solution with a small amount of alcohol, prepare 1% DES solution with pH 7.0 phosphate buffer solution, take 2ml of fresh bacterial solution and 2ml of 1% DES, mix well, shake in the dark at 30°C, 200rpm, respectively After treatment for 10, 20, 30, 40, 60, and 80 minutes, add 2ml of 1% sodium thiosulfate to terminate the reaction, serially dilute four tim...

Embodiment 2

[0036] Cultivation of QY131 Yeast

[0037] The slant medium is malt juice agar medium, and the shake flask medium formula is molasses 30g / L, glucose 10g / L, magnesium sulfate 1.2g / L, ammonium sulfate 1.2g / L, urea 0.2g / L, potassium dihydrogen phosphate 1.2g / L, zinc sulfate 0.2g / L, add water to dissolve, adjust the pH to 5.4 (±0.1), and finally adjust the volume to 1L. Use an inoculation needle to pick a ring of slant strains and insert it into a 500ml shake flask containing 100ml of medium, at 28°C, with a rotation speed of 250rpm, and shake it for 16 hours, then insert it into 2000ml of medium (the formula is the same as that of the shake flask medium) ) in a 5L automatic fermenter with a temperature of 28°C, a stirring speed of 300rpm, and an air volume of 0.5vvm for 20 hours. Then it is centrifuged to collect the bacteria, and the bacteria can be used for CMP and phosphorylcholine biotransformation of citicoline after the bacteria are air-dried or quick-frozen to break the w...

Embodiment 3

[0039] Biotransformation of Citicoline in Shake Flasks

[0040] Add 100mL reaction solution into 250ml shake flask, which contains CMP25mmol / L, phosphate buffer 200mmol / L, glucose 4g, phosphorylcholine 5g, magnesium sulfate 0.4g, QY131 yeast 5g (dry weight). 32°C, rotation speed 150rpm, shaking reaction for 6-8h. Feed 2g of glucose every 2 hours, depending on the reaction situation, consider adding sugar 2-3 times, and measure the conversion rate every 2 hours. After reacting for 6 hours under the above conditions, the conversion rate of citicoline can reach 83%.

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Abstract

The invention belongs to the field of biological medicine and relates to S. cerevisiae strain for producing citicoline through bioconversion and application of the S. cerevisiae strain, namely the S. cerevisiae strain of high activity CMP (cytidine monophosphate) kinase and high activity phosphoryl choline-cytidyltransferase and the application of the S. cerevisiae strain in the production of citicoline through bioconversion. The S. cerevisiae strain provided by the invention has a classification name of S. cerevisiae, and has the Preservation No. of CGMCC7978 in CGMCC (China General Microbiological Culture Collection Center).

Description

technical field [0001] The invention belongs to the field of biomedicine. It involves the selection and breeding of a strain of biotransformation citidyle, that is, a strain of Saccharomyces cerevisiae (S. Application of citicoline. Background technique [0002] The manufacture of citicoline currently adopts three methods: chemical synthesis, double-bacteria fermentation and yeast biotransformation. Due to the high cost of chemical synthesis and serious environmental pollution, only Italy uses this method to produce a small amount. After the 1990s, Yamayama in Japan mainly used the yeast biotransformation method for production. However, due to the high price of the main raw material CMP, Japan developed a dual-bacteria fermentation method after 2000. It utilizes genetically engineered bacteria such as Escherichia coli and Corynebacterium ammoniagens. Using orotic acid (or uracil) as the main raw material to produce citicoline, the conversion rate is 56%. The yeast biotr...

Claims

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Application Information

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IPC IPC(8): C12N1/18C12P19/30C12R1/865
Inventor 邱蔚然周长林王鹏飞邱志云周洁马志娟
Owner NANTONG QIUZHIYOU BIOSCI & BIOTECH
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