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An Escherichia coli engineering bacterium for producing 2,3,5,6-tetramethylpyrazine and its application

A technology of tetramethylpyrazine and Escherichia coli, applied in the direction of microorganism-based methods, bacteria, microorganisms, etc., can solve the problems of many by-products and high requirements for reaction conditions, and achieve less reaction by-products, low production equipment requirements, The effect of simplifying the production process

Active Publication Date: 2020-05-15
BEIJING TECHNOLOGY AND BUSINESS UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above-mentioned chemical synthesis methods all have the unfavorable factors of high requirements on reaction conditions and more by-products

Method used

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  • An Escherichia coli engineering bacterium for producing 2,3,5,6-tetramethylpyrazine and its application
  • An Escherichia coli engineering bacterium for producing 2,3,5,6-tetramethylpyrazine and its application
  • An Escherichia coli engineering bacterium for producing 2,3,5,6-tetramethylpyrazine and its application

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Experimental program
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Embodiment 1

[0035] Embodiment 1: Construction of Escherichia coli engineering bacteria E.coli BL15

[0036] 1. Gene synthesis

[0037] Refer to the acetoin anabolic operon alsRSD (NCBI gene number: A3UG_05855, A3UG_05860, A3UG_05865) derived from Enterobacter cloacae subsp.dissolvens SDM published by NCBI, the NADH oxidase gene nox (NCBI gene number: AF536177.1) and P tac The sequence of the promoter (NCBI number: X15234.1), the corresponding double-stranded DNA is artificially synthesized. Endonuclease cutting sites BglII and XhoI were added to the 5' end and 3' end of alsRSD respectively, and endonuclease cutting site BglII was added to the 3' end of gene nox.

[0038] 2. The acetoin synthetic metabolic operon alsRSD was inserted into the plasmid pET-28a(+)

[0039] The artificially synthesized operon alsRSD was digested by BglII and XhoI, and the pET-28a(+) expression plasmid was treated by BglII and XhoI double digestion, the gel was cut and recovered, and then the expression plasm...

Embodiment 2

[0042] Embodiment 2: prepare the nutrient solution of Escherichia coli engineering bacteria E.coli BL15

[0043] 1. Plate culture: Aseptically streak E. coli engineering bacteria E.coli BL15 onto an agar plate (LB medium contains 1.5-2.0% agar and 50mg / L kanamycin sulfate), in a constant temperature incubator at 37 ± 1 Incubate at ℃ for 12±2 hours;

[0044] 2. Seed culture: Aseptic operation, use a sterile toothpick to pick up a single colony from step (1), inoculate it into LB liquid medium containing 50 mg / L kanamycin sulfate, and culture in a constant temperature shaker at 37±1°C with shaking After 12±2 hours, the activated bacterial liquid of Escherichia coli engineering bacteria E.coli BL15 was obtained;

[0045] Wherein, the formula of the LB medium described in the above steps (1) and (2) is: 1L of distilled water contains: 5g of yeast powder, 10g of peptone, 10g of sodium chloride, and sterilized at 121° C. for 20 minutes.

Embodiment 3

[0046] Embodiment 3: Escherichia coli engineering bacteria E.coli BL15 ferments glucose to produce acetoin

[0047] Under sterile conditions, activate Escherichia coli engineering bacteria E.coli BL15, and inoculate the activated bacterial solution into the fermentation medium with an inoculation volume of 5%-10% by volume. The temperature is 37±1°C, the stirring speed is 200-500rpm, and the sterile air intake is 0.5vvm-2.0vvm, and the fermentation is carried out for 48-96 hours. During the cultivation process, when the glucose concentration in the medium drops below 20g / L, add sterile 600-800g / L glucose solution until the glucose concentration in the medium is 40-60g / L. When the alcohol concentration no longer increases, stop the fermentation.

[0048] Wherein, the formula of the above-mentioned fermentation medium is: 1L distilled water contains 80g glucose, 10g yeast powder, 3.0gCH 3 COONa·3H 2 O, 0.60g KH 2 PO 4 , 0.40g MgSO 4 , 0.10g MnSO 4 , 0.060g FeSO 4 .

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Abstract

The invention discloses an escherichia coli engineering bacterium for efficiently producing 2,3,5,6-tetramethylpyrazine. The engineering bacterium is an escherichia coli carrying an acetoin syntheticoperator alsRSD from Enterobacter cloacae and an NADH oxidase gene nox from Lactobacillus brevis. Using the genetic engineering bacterium provided by the invention to transform glucose to produce 2,3,5,6-tetramethylpyrazine, the highest yield is up to 16.40g / L, the purity of a product is more than or equal to 98%, and the engineering bacterium has the advantages of mild reaction conditions, few by-products, and easy separation and purification of the product, and has a very good application value.

Description

technical field [0001] The invention relates to an Escherichia coli genetic engineering bacterium for producing 2,3,5,6-tetramethylpyrazine and its application. Background technique [0002] 2,3,5,6-tetramethylpyrazine (2,3,5,6-Tetramethylpyrazine, TMP) is an effective drug for treating cardiovascular and cerebrovascular diseases. Due to its remarkable curative effect, TMP is highly respected by doctors and patients, and has become one of the most commonly used drugs in clinical practice. The demand for it is huge, so the research on the synthesis of TMP has attracted widespread attention. At present, the synthesis methods of TMP are mainly chemical methods, including gas-phase catalytic method, α-aminoketone condensation method, o-diamine and o-diketone condensation method, and nitrogen-substituted o-diketone or α-azido-ketone condensation method (Zang Chuan Near, et al. Shandong Chemical Industry. 2015, 44(18): 23-25). However, the above-mentioned chemical synthesis meth...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12P17/12C12R1/19
CPCC12N9/0036C12N9/1022C12N9/88C12N15/52C12P17/12C12Y106/99003C12Y202/01006C12Y401/01005
Inventor 李秀婷徐友强孙宝国许春艳
Owner BEIJING TECHNOLOGY AND BUSINESS UNIVERSITY
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