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Method for producing 5 '-cytidine monophosphate

A cytidine and uridine kinase technology, applied in the fields of genetic engineering and microbial engineering, can solve the problem of low yield and achieve the effects of enhanced transformation, favorable separation and purification, and high concentration

Active Publication Date: 2022-03-01
江苏香地化学有限公司 +1
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

However, this method needs to crush the recombinant bacteria and then separate the enzyme, and the yield is not high

Method used

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  • Method for producing 5 '-cytidine monophosphate
  • Method for producing 5 '-cytidine monophosphate
  • Method for producing 5 '-cytidine monophosphate

Examples

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Embodiment 1

[0027] Embodiment 1: The construction method of the recombinant Escherichia coli that is used to produce 5'-cytidylic acid

[0028] (1) Construction of recombinant plasmid pRSFDuet- for overexpression of uridine kinase gene udk

[0029] Using primers udk-FW: CATGCCATGGGCactgatcagtctcatcagtg (SEQ ID NO: 7) and udk-RS: CGGGATCCTTATTCAAAGAACTGACTT (SEQ ID NO: 8), the E. coli genome was used as a template for PCR amplification to obtain udk The target fragment (the nucleotide sequence is shown in SEQ ID NO: 5, and the amino acid sequence of the encoded enzyme is shown in SEQ ID NO: 6). PCR amplification conditions: pre-denaturation at 98°C for 5 min; denaturation at 95°C for 30 s; annealing at 55°C for 30 s; extension at 72°C for 1 min; set cycle 29 times; extension at 72°C for 10 min; final extension at 16°C insulation. PCR amplification system: 5×PSbuffer 20 μL, dNTP 10 μL, upstream / downstream primers (10 μmol L -1 ) 2 μL, template 1 μL, enzyme 1 μL, water 66 μL. restrict...

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Abstract

The invention discloses a method for producing 5 '-cytidine monophosphate, and belongs to the field of genetic engineering and microbial engineering. A cytidine monophosphate pyrophosphorylase gene ushA and a nucleotide 5 '-monophosphate nucleosidase gene ppnN of escherichia coli are knocked out through a gene knockout technology, and the escherichia coli mutant capable of efficiently accumulating 5'-cytidine monophosphate (5 '-CMP) is obtained. The recombinant Escherichia coli capable of efficiently producing 5 '-CMP is further obtained by overexpressing a uridine kinase gene udk, and the fermentation level of the 5'-CMP can reach 31.8 g / L. The efficient Escherichia coli strain can be used for realizing industrial production of the 5 '-CMP, and is beneficial to reducing the production cost and environmental pollution and realizing green biological manufacturing.

Description

technical field [0001] The invention relates to a method for producing 5'-cytidylic acid, belonging to the fields of genetic engineering and microbial engineering. Background technique [0002] Nucleotides are mainly involved in the formation of nucleic acids and have important biological functions. Nucleotides and their derivatives can be widely used in agricultural production, food, medicine and other fields. The chemical synthesis of nucleotides usually uses phosphorus oxychloride as a phosphate donor, and the nucleosides are directly phosphorylated to produce the corresponding nucleotides. However, when this chemical method is used to synthesize 5'-cytidylic acid, it is necessary to add a protecting group to the 2', 3'-hydroxyl of ribose, and then carry out the phosphorylation reaction. The chemical synthesis of nucleotides has many operating steps, long routes, poor stereoselectivity, expensive and toxic reagents, poor operability, and high production costs, and is no...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P19/30C12R1/19C12R1/125
CPCC12N9/1241C12N9/1205C12N9/16C12P19/305C12Y207/01048C12Y301/03005Y02P20/55
Inventor 杨海泉张玮琪张飞龙梁国斌张剑陈献忠董田田
Owner 江苏香地化学有限公司
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