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Tetrahydromethylpyrimidine carboxylic acid biosynthetic gene and application thereof

A tetrahydromethylpyrimidine carboxylic acid and biosynthesis technology, applied in the field of genetic engineering and compound biotechnology production, can solve the problems of poor stereospecificity, difficult separation and extraction, and high cost

Active Publication Date: 2021-02-09
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

Although tetrahydromethylpyrimidinecarboxylic acid is easy to be chemically synthesized, the cost of its chemical synthesis and production is very high due to the high price of its precursor substances (such as diaminobutyric acid, etc.)
In view of the difficulties in downstream separation and extraction caused by (1) severe equipment corrosion, (2) discontinuous production, and (3) production of structural analogues in the fermentation process of halophilic microorganisms and the chemical synthesis of tetrahydromethylpyrimidinecarboxylic acid There are problems such as (1) complicated process, (2) poor stereospecificity, and (3) high cost. It is necessary in this field to develop a method that is not regulated by salt concentration and nitrogen source concentration and can produce high-intensity at low salt concentration. Tetrahydromethyl pyrimidine carboxylic acid recombinant strain

Method used

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  • Tetrahydromethylpyrimidine carboxylic acid biosynthetic gene and application thereof
  • Tetrahydromethylpyrimidine carboxylic acid biosynthetic gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: Codon optimization and total gene synthesis of tetrahydromethylpyrimidinecarboxylic acid biosynthesis gene

[0053] The present invention is based on diaminobutyric acid-2-oxoglutarate aminotransferase (EC 2.6.1.76) and L-2,4-diaminobutyric acid transacetylase (EC 2.3.1.178) derived from Halomonas elongatus and tetrahydromethylpyrimidinecarboxylate synthase (EC 4.2.1.108) (the ID numbers of the three enzymes in the protein database are O52250, O52249 and O52251 respectively), using the degeneracy of codons to encode the above four Design and optimize the codon preference, GC content, and spatial structure of the mRNA of the hydromethylpyrimidine carboxylic acid biosynthetic enzyme, entrust Sangon Bioengineering (Shanghai) Co., Ltd. to carry out the whole gene synthesis, and obtain the nucleotide sequence The polynucleotide encoding diaminobutyrate-2-oxoglutarate aminotransferase as shown in SEQ ID NO: 1, the nucleotide sequence as shown in SEQ ID NO: 2 encodi...

Embodiment 2

[0054] Example 2: Construction of tetrahydromethylpyrimidinecarboxylic acid biosynthetic gene expression vector

[0055] (1) Synthesize two primers respectively having the nucleotide sequences shown in SEQ ID NO: 7 and SEQ ID NO: 8 in the sequence listing. Nucleotide sequence such as SEQ ID NO: 7 and the 5'-end of the primer shown in SEQ ID NO: 8 are set respectively Nde I and xho I Restriction sites and their protective bases. Using the polynucleotide whose nucleotide sequence is shown in SEQ ID NO: 1 as a template, the polymerase chain reaction (PCR) was carried out using the above two primers SEQ ID NO: 7 and SEQ ID NO: 8. The DNA polymerase is Phanta from Nanjing Novizan Biotechnology Co., Ltd. ® Super-Fidelity DNA polymerase. The PCR amplification program was: 95°C for 5min; 94°C for 45s, 56°C for 45s, 72°C for 2min, a total of 30 cycles; 72°C for 10min, then drop to 10°C. PCR products were detected by agarose gel electrophoresis. Under the irradiation of ultrav...

Embodiment 3

[0058] Example 3: Construction of tetrahydromethylpyrimidinecarboxylic acid biosynthetic gene cluster

[0059] 3.1. Tetrahydromethylpyrimidinecarboxylic acid biosynthesis gene cluster P(T7)- lacO - Construction of RBS-2-RBS-1-RBS-3-Ter (T7)

[0060] (1) Synthesize two primers respectively having the nucleotide sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14 in the sequence listing. Nucleotide sequences such as SEQ ID NO: 13 and the 5'-ends of the primers shown in SEQ ID NO: 14 are set respectively Nhe I and Speech I Restriction sites and their protective bases. The expression vector pECT-1 was used as template for PCR amplification. The PCR amplification procedure is the same as (1) in Example 2. The PCR amplified products were detected by agarose gel electrophoresis, separated, and recovered by gel cutting. Nhe I and Speech I double enzyme digestion, using T4 DNA ligase (purchased from Bao Biological Engineering (Dalian) Co., Ltd. (TaKaRa)) to connect the sam...

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Abstract

The invention discloses a tetrahydromethylpyrimidine carboxylic acid biosynthetic gene and an application thereof, and belongs to the field of genetic engineering and compound biotechnology production. The tetrahydromethylpyrimidine carboxylic acid biosynthetic gene disclosed by the invention is artificially synthesized polynucleotide optimized by codon, mRNA space structures and the like, and canbe connected with various gene expression cassettes in an operable manner to obtain expression constructs and recombinant cells, which have adjustable gene expression quantity and are suitable for various hosts. The obtained recombinant cell can be used for producing tetrahydromethylpyrimidine carboxylic acid by taking aspartic acid as a precursor, and can also be used for biotransformation and fermentation production of the tetrahydromethylpyrimidine carboxylic acid by taking carbohydrate such as glucose as a carbon source under the condition that the total salt concentration is as low as 0.5% (m / v), and the yield can be as high as 30g / L. Therefore, the tetrahydromethylpyrimidine carboxylic acid biosynthetic gene and gene clusters, the expression constructs and the recombinant cells thereof provided by the invention have extremely high application value.

Description

technical field [0001] The invention relates to the fields of genetic engineering and compound biotechnology production, more specifically, the invention relates to tetrahydromethylpyrimidine carboxylic acid biosynthesis gene and application thereof. Background technique [0002] Tetrahydromethylpyrimidinecarboxylic acid is a natural cyclic amino acid. Tetrahydromethylpyrimidinecarboxylic acid is also a natural compatible solute, which can be used as an osmotic pressure regulator to help cells maintain osmotic balance and adapt cells to a salt environment. Tetrahydromethylpyrimidinecarboxylic acid is also an excellent compound with stabilizing effect. It can be used as a protective agent for biomolecules (such as proteins, nucleic acids, etc.), stabilize protein molecules, maintain enzyme activity, and promote the folding of polypeptide chains into active The protein can reduce the adverse effects of freezing, drought, high temperature and other harsh conditions on protein....

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N9/88C12N15/70C12N1/21C12P19/38C12R1/19
CPCC12N9/1029C12N9/1096C12N9/88C12N15/70C12P17/12C12Y203/01178C12Y206/01076C12Y402/01108
Inventor 汪仁李宜奎钱彬彬李洁李杨
Owner INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
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