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Recombinant escherichia coli for producing 5-methylpyrazine-2-carboxylic acid

A technology for recombining Escherichia coli and methylpyrazine, applied in the field of genetic engineering, can solve the problems of unstable industrial production of recombinant plasmids, short transformation time, environmental pollution, etc., and achieve good industrialization prospects, shorten the cycle, and high transformation time.

Active Publication Date: 2020-01-14
迪嘉药业集团股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The conversion time of this method is relatively short, and the molar conversion rate is high, but the yield of this method is relatively low, and the recombinant plasmid is unstable in industrial production, and there are environmental pollution problems caused by IPTG, antibiotics, etc.

Method used

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  • Recombinant escherichia coli for producing 5-methylpyrazine-2-carboxylic acid
  • Recombinant escherichia coli for producing 5-methylpyrazine-2-carboxylic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1. Construction of recombinant Escherichia coli producing 5-methylpyrazine-2-carboxylic acid

[0049] According to the NCBI published E. coli Escherichia coli The genome sequence of BL21(DE3) designed the amplification primers for the upstream and downstream homology arms of the flhD site, and the upstream and downstream primers for the left arm were:

[0050] up-flhD-F:5'-CGTCTTTTTTACTGCCCGGGATG-3'

[0051] up-flhD-R:5'-ATCTTAAGGGTCTGATCACATTATACGAGCCGATGATTAATTGTCAAAGCAAATTCATACCGGCATCATGC-3'

[0052] The upstream and downstream primers of the right arm are:

[0053] B-down-flhD-F:5'-CGGTACTCCGGATTGGTTGAGTGTTTCAGCAACTCGGAGGTATGC-3'

[0054] down-flhD-R:5'-CTGATTCTCCGCATTGAACAAGTGG-3'

[0055] Escherichia coli Escherichia coli The left and right arms contained in the amplified integration frame in the BL21(DE3) genome.

[0056] According to the sequence of xylene monooxygenase gene Gene ID: 1218754 and Gene ID: 1218743, benzyl alcohol dehydrogenase gene...

Embodiment 2

[0061] Example 2. Construction of different xylene monooxygenase double subunit genes wxya with xylA 5-Methylpyrazine-2-carboxylic acid-producing recombinant Escherichia coli with copy number ratio

[0062] to build E. coli MPCA2 (xylM:xylA=2:1), E. coli MPCA3 (xylM:xylA=1:2) and E. coli MPCA4 (xylM:xylA=2:2) according to NCBI published Escherichia coli Escherichia coli The genome sequence of BL21(DE3) designed the upstream and downstream homologous arm amplification primers of the motA site. The left arm primers are all consistent, and the left arm upstream and downstream primers are respectively:

[0063] up-motA-F:5'-CGATGTTCGGCTGCTTATTCACTTC-3'

[0064] up-motA-R:5'-ATCTTAAGGGTCTGATCACATTATACGAGCCGATGATTAATTGTCAAACTTTCCTCGGCATTTTATTGGCTTAC-3'

[0065] E. coli The right arm primer of MPCA2 (xylM:xylA=2:1) ​​is:

[0066] M-down-motA-F: 5’-GGTGGCTAGCATTTGAGATTTCGCCATCAACCGAT-3’

[0067] down-motA-R:5'-TTCCAGCTGCAACTGCTGC-3'

[0068] E. coli MPCA3 (xyl...

Embodiment 3

[0122] Example 3. Production of 5-methylpyrazine-2-carboxylic acid by fermenting recombinant Escherichia coli

[0123] The obtained nine 5-methylpyrazine-2 carboxylic acid-producing recombinant Escherichia coli MPCA1-9 were cultured with seed medium at 37°C and 220rpm for 12h, and then transferred to fermentation culture with a 1% inoculation amount based on 37 ℃, 220rpm fermentation for 12h, centrifuged to collect the cells, and then adding 12g / L substrate DMP in pH 8 buffer for catalysis for 36h. The formula of the buffer solution is: 1.6 g / L sodium dihydrogen phosphate dihydrate, 67.8 g / L disodium hydrogen phosphate dodecahydrate. The 9 strains of above-mentioned construction are carried out fermentation output comparison, when catalysis ends, use the content of 5-methylpyrazine-2-carboxylic acid in the fermented broth to measure by high performance liquid chromatography, the result is as follows figure 2 shown. in E. coli MPCA8 (xylM:xylA=2:3) had the highest yield, ...

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Abstract

The invention relates to recombinant escherichia coli for producing 5-methylpyrazine-2-carboxylic acid, in particular to a method for increasing 5-methylpyrazine-2-carboxylic acid by adjusting the proportion of dimethylbenzene monooxygenase double subunits. According to the adjustment of the proportion of the dimethylbenzene monooxygenase double subunits, escherichia coli (E.coli BL21 (DE3) is used as an original strain, a 5-methylpyrazine-2-carboxylic acid synthetic route is constructed by utilizing CRISPR / cas9 homologous recombination to integrate xylene monooxygenase, benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase genes. According to the strain disclosed by the invention, the yield of the 5-methylpyrazine-2-carboxylic acid in recombinant escherichia coli can be increased to15.6 g / L, and a foundation is laid for industrial production of the 5-methylpyrazine-2-carboxylic acid.

Description

technical field [0001] The invention relates to a recombinant Escherichia coli producing 5-methylpyrazine-2-carboxylic acid, and also relates to a method for increasing the ratio of xylene monooxygenase disubunits to increase 5-methylpyrazine-2-carboxylic acid, which belongs to The field of genetic engineering technology. Background technique [0002] 5-Methylpyrazine-2-carboxylic acid (5-Methylpyrazine-2-carboxylic acid, MPCA) is used in the synthesis of glipizide, acipimus and methyl 5-methylpyrazine-2-carboxylate It is an important intermediate of drugs such as and used as a metal complex for the preparation of catalysts. At present, its synthesis mainly adopts chemical synthesis method, which can be divided into intermolecular ring method, pyrazine side chain multi-step synthesis method, direct oxidation method and electrochemical method. However, the chemical synthesis method has the problems of high requirements on reaction conditions, the use of a large amount of ox...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P17/12C12R1/19
CPCC12N9/0006C12N9/0071C12N9/0067C12N15/70C12P17/12
Inventor 李广生杨涛王祥法门晋名曲欣欣王晓群李庆猛
Owner 迪嘉药业集团股份有限公司
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