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Overexpressed phosphorylcholine cytidine transferase saccharomyces cerevisia genetically engineered bacteria as well as construction method and application thereof

A technology of genetically engineered bacteria and phosphorylcholine, applied in the field of genetic engineering, can solve problems such as unsuitability for large-scale production, difficult control of yeast quality, and impact on biotransformation rate, and achieve convenient and fast biotransformation process, increase permeability, The effect of high yield

Inactive Publication Date: 2017-12-19
NANTONG QIUZHIYOU BIOSCI & BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But the disadvantage is that it is not suitable for large-scale production, the quality of each batch of yeast is difficult to control, and the storage and processing environment of yeast has a great impact on the biotransformation rate
Another example: the defect of Chinese patent CN103436455A is that the yield of fermentable sugar by yeast solids is about 25%-30%, and the relative cost of bacteria is relatively high

Method used

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  • Overexpressed phosphorylcholine cytidine transferase saccharomyces cerevisia genetically engineered bacteria as well as construction method and application thereof
  • Overexpressed phosphorylcholine cytidine transferase saccharomyces cerevisia genetically engineered bacteria as well as construction method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0029] Embodiment 1: Construction of recombinant bacteria

[0030] According to the Saccharomyces cerevisiae encoding phosphorylcholine cytidine transferase gene (SEQ ID NO: 1) in the Genbank database, the primers cct-S and cct-A (SEQ ID NO: 2~3) were designed and synthesized, both provided by Kings Rui Technology (Nanjing) Co., Ltd. conducts total gene synthesis.

[0031] A fragment with a length of 1277bp was obtained, and the fragment was purified by nucleic acid electrophoresis.

[0032] After the fragment was recovered, it was connected to the pYES2.0-Kanmx vector (preserved in the Department of Microbiology, China Pharmaceutical University), and the enzyme-digested product was constructed to obtain pYES2.0-Kanmx- cct Recombinant plasmid, the measured total sequence length is 8224bp see attached figure 1 .

[0033] Digest pYES2.0-Kanmx- with Kpn I and BamH I cct Recombined plasmids were digested and verified by agarose gel electrophoresis, and the results of nucleic ...

Embodiment 2

[0040]Example 2: Enrichment and expression of genetically engineered bacteria

[0041] (1) Inoculate the genetically engineered bacteria QZ-016 from the YPD-resistant plate of G418 into 5mL YPD liquid medium and cultivate to OD 600 =0.6.

[0042] (2) Add D-galactose with a final concentration of 2% to the above-mentioned culture medium, induce for 16 hours, and obtain the primary seed culture medium.

[0043] (3) Inoculate the seed culture solution obtained in step (2) into 100mL CMP-YPD liquid medium, culture on a shaker for 16 hours, add D-galactose with a final concentration of 2%, induce for 8 hours, and obtain a secondary seed culture solution .

[0044] (4) Inoculate the seed culture solution obtained in step (3) into 500mL YPD liquid medium, culture on a shaker for 16 hours, add D-galactose at a final concentration of 2%, and induce for 8 hours to obtain a tertiary seed culture solution.

[0045] (5) Inoculate the seed culture solution obtained in step (4) into 18L Y...

Embodiment 3

[0060] Embodiment 3: the air-drying of yeast strain is standby:

[0061] The wet cells of the genetically engineered bacteria obtained from the above fermentation were collected and dried in a vacuum oven at 40°C. Turn and grind once every 2 hours until uniform powdery particles are obtained, and place in a desiccator for later use.

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Abstract

The invention relates to overexpressed phosphorylcholine cytidine transferase saccharomyces cerevisia genetically engineered bacteria as well as a construction method and application thereof. The construction method comprises the steps of cloning saccharomyces cerevisia sourced gene cct capable of encoding phosphorylcholine cytidine transferase to a pYES2.0-Kanmx carrier so as to construct recombinant plasmid pYES2.0-Kanmx-cct, and transferring the recombinant plasmid pYES2.0-Kanmx-cct into S.cerevisiae HG, so as to obtain the saccharomyces cerevisia genetically engineered bacteria. The yield of a solid matter of the strain to fermentable sugar is 50%-56%, the strain can be used for manufacturing a citicoline product from 5'-cytidine monophosphate and phosphorylcholine through bioconversion, and after the strain reacts for 7 hours, the molar conversion ratio reaches up to 73%. The yield of the strain in a fermentation system for producing and culturing the strain is high. When applied to the production of citicoline, the strain has the advantages of low cost and manufacturing energy consumption, short reaction period and the like.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a genetically engineered bacterium of Saccharomyces cerevisiae overexpressing phosphorylcholine cytidine transferase, its construction method and application. Background technique [0002] Citicoline (CDP-C) belongs to nucleic acid biochemical drugs. The Chinese Pharmacopoeia 2005 edition named it Citicoline. Its chemical name is choline cytosine nucleoside-5'-diphosphate monosodium salt, white Crystalline powder. Citicoline is an intermediate in the biosynthesis of lecithin. By improving the metabolism of phospholipids, it can improve the metabolism of brain tissue and regulate the tension of cerebral blood vessels. It is the main drug for treating brain disturbance in brain surgery and psychiatry. It has a unique curative effect on the disturbance of consciousness caused by trauma and brain surgery, as well as some chronic diseases such as hemiplegia, m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/54C12N15/81C12P19/30C12R1/865
CPCC12N9/1241C12P19/305C12Y207/07015
Inventor 邓童心邱蔚然周长林季晨明莫违邱志云
Owner NANTONG QIUZHIYOU BIOSCI & BIOTECH
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