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Codon optimized phosphocholine cytidylyltransferase gene and expression thereof

A technology of codon optimization and phosphorylcholine, applied in the field of microorganisms, can solve the problems of low conversion rate, cumbersome process and high production cost, and achieve the effect of high crude enzyme liquid enzyme activity

Active Publication Date: 2015-11-11
INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chemical synthesis usually needs to prepare some intermediates first, the process is cumbersome and the production cost is too high, the operation requirements are strict, and the conversion rate is not high

Method used

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  • Codon optimized phosphocholine cytidylyltransferase gene and expression thereof
  • Codon optimized phosphocholine cytidylyltransferase gene and expression thereof
  • Codon optimized phosphocholine cytidylyltransferase gene and expression thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Example 1 Construction of recombinant Escherichia coli.

[0026] 1.1 Codon optimization Codon optimization and whole gene synthesis of phosphorylcholine cytidine transferase gene.

[0027] The present invention is based on the phosphorylcholine cytidine transferase derived from Saccharomyces cerevisiae cct The gene sequence adopts the method of whole gene synthesis to optimize the phosphorylcholine cytidine transferase gene according to the preferred codon of Escherichia coli, and entrusts Nanjing Tuoda Biotechnology Co., Ltd. to synthesize it. The pUC57 plasmid is used as a subcloning vector in the gene synthesis service for Phosphocholine cytidine transferase gene The open reading frame of the phosphorylcholine cytidine transferase gene synthesized by codon-optimized preference in Escherichia coli is 1275 bases. The homology comparison was carried out by VectorNTI software, and it was found that the homology between the synthetic sequence and the original sequence wa...

Embodiment 2

[0034] Example 2 Inducible expression of codon-optimized phosphorylcholine cytidine transferase.

[0035] 2.1 Induced expression of recombinant bacteria BL21(DE3) / pET-29a-cct-op and Rosetta(DE3) / pET-29a-cct-op.

[0036] Strain activation: Streak the recombinant bacteria L21(DE3) / pET-29a-cct-op and Rosetta(DE3) / pET-29a-cct-op on the solid plate containing kanamycin LB on the clean bench A single colony was isolated and cultured overnight at 37°C in a constant temperature incubator.

[0037]Seed culture: Under sterile conditions, pick a single colony on the plate, insert it into a test tube of 3 mL of LB liquid medium added with kanamycin, and culture it on a shaker at 37°C for 12 hours at a constant temperature of 200rpm.

[0038] Induced expression: Transfer the seed solution to a 50ml Erlenmeyer flask (250ml) containing LB medium containing kanamycin at a 2% (v / v) inoculation amount. Proliferate at 220 rpm at 37°C for 2.5-3h to OD 600 To 0.6-0.8, add IPTG at a final concen...

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Abstract

The invention relates to a codon optimized phosphocholine cytidylyltransferase cct gene and expression thereof, wherein the cct gene is constructed on the basis of escherichia coli codon preference. The optimized cct gene is obtained through a total synthesis method, the nucleotide sequence of the gene is represented by SEQ ID No.1, and the sequence of coding amino acid is represented by SEQ ID No.2. The length of the gene is 1275 bp, and encodes 424 amino acids. The homology between optimized cct gene and wild cct gene is 76.6%; the optimized cct gene is connected to a carrier pET-29a(+) to obtain a recombinant carrier pET-29a-cct-op, and the recombinant carrier is converted to escherichia coli. After IPTG inducible expression and enzyme activity detection, an engineering bacterium strain, which has been modified by preference and can efficiently express phosphocholine cytidylyltransferase, is obtained. The invention has the advantages that brewer's yeast cct gene is redesigned according to the preference of escherichia coli codon so as to improve the efficiency of escherichia coli on expressing phosphocholine cytidylyltransferase, the gene can be used to produce phosphocholine, the industrial output can be increased, and the economic profits for enterprises are increased.

Description

technical field [0001] The invention belongs to the technical field of microbes, and in particular relates to a gene and expression thereof of phosphorylcholine cytidine transferase (Cholinephosphatecytidylyltransferase, EC2.7.7.15, CCT) optimized according to the preferred codons of Escherichia coli. Background technique [0002] Citicoline (cytidine-5'-diphosphatecholine) is an important nucleic acid derivative and the main coenzyme of lecithin biosynthesis, which can promote brain cell respiration, restore nerve tissue function, improve brain metabolism and circulation, and can be used for treatment Cerebrovascular disease, Parkinson's syndrome, and depression have obvious curative effects on the treatment of neurological diseases caused by craniocerebral injury and cerebrovascular accident. It also has a certain curative effect on delaying aging, improving learning effect and memory, so it is widely used clinically. [0003] There are many reports on the synthesis of ci...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N15/70C12N1/21C12R1/19
CPCC12N9/1241
Inventor 李晓丹汪仁夏冰
Owner INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
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