A method of producing ectoine by high-density culture of recombinant escherichia coli

A technology for recombining Escherichia coli and ectoine, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve problems such as increasing the difficulty of downstream purification processes, equipment corrosion, and complex components of fermentation products

Active Publication Date: 2015-05-06
南京众惠生物材料科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The use of high-salt medium in this method is easy to cause corrosion to the equipment, and the fermentation product has complex components, which increases the difficulty of the downstream purification process, makes the production cost of ectoine remain high, and seriously restricts the large-scale application of ectoine
[0004] The existing production methods severely restrict the industrial production and large-scale application of ectoine, so it is of great practical significance to develop an efficient ectoine production method to reduce its production cost.

Method used

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  • A method of producing ectoine by high-density culture of recombinant escherichia coli
  • A method of producing ectoine by high-density culture of recombinant escherichia coli
  • A method of producing ectoine by high-density culture of recombinant escherichia coli

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1, Construction of Escherichia coli ectoine high-yield strain BW-pBAD-ectABC

[0070] (1) Expression of ectoine synthesis gene cluster EctABC in Escherichia coli

[0071] 1. PCR amplification of the coding sequence of the ectoine synthesis gene cluster EctABC

[0072] Using the genomic DNA of Halomonas elongate (CGMCC No.1.6329) as a template, PCR amplification was performed with primers EctABC-P1 and EctABC-P2 to obtain PCR amplification products.

[0073] EctABC-P1: 5'-CCTA GCTAGC ATGAACGCAACCACAGAGCCCTTTA-3'

[0074] EctABC-P2: 5'-CCG CTGCAG TTACAGCGGCTTCTGGTCGTCGGCT-3'

[0075] 2. Digestion and ligation

[0076] The PCR amplified product was double-digested with NheI and PstI, and ligated with the pBAD / HisA plasmid large fragment previously cut with NheI and PstI to obtain a recombinant plasmid.

[0077] 3. Transformation, screening and sequence verification

[0078] Transform the recombinant plasmid prepared above into Escherichia coli DH5α by calc...

Embodiment 2

[0081] Embodiment 2, the expression of EctABC gene in Escherichia coli ectoine high-yield strain BW-pBAD-ectABC

[0082] Pick a single colony of BW-pBAD-ectABC and inoculate it into 5 ml of LB medium containing ampicillin (100 μg / ml), and culture overnight at 37°C. Inoculate 1ml of the overnight culture into 100ml of LB medium containing ampicillin (100μg / ml) and culture at 37°C with vigorous shaking (200rpm) to the OD of the fermentation broth 600 When the value reaches about 0.6-0.8, add L-arabinose to the fermentation system (the final concentration of L-arabinose is 1g / L), and continue to cultivate at 30°C for 6 hours. Escherichia coli BW-pBAD containing empty vector was set as negative control.

[0083] After fermentation, centrifuge at 5000rpm for 15 minutes to collect the bacteria; resuspend the bacteria in PBS buffer with pH 7.0, and centrifuge at 12,000rpm for 15min after ultrasonication. Collect the supernatant, which is the crude enzyme solution containing the tar...

Embodiment 3

[0084] Example 3, the application of the strain BW-pBAD-ectABC with better ectoine synthesis ability and the preservation number is CGMCC NO.8334

[0085] (1) Preparation of ectoine by strain fermentation and biotransformation

[0086] 1. Strain fermentation and expression of EctABC gene

[0087] Pick a single colony of BW-pBAD-ectABC and insert it into 20ml of LB medium containing ampicillin (100μg / ml), and culture overnight at 37°C. Inoculate 5ml of the overnight culture into 500ml LB medium containing ampicillin (100μg / ml) and culture at 37°C with vigorous shaking (200rpm) to the OD of the fermentation broth 600 When the value reaches about 0.6-0.8, add L-arabinose to the fermentation system (the final concentration of L-arabinose is 1g / L), continue to cultivate at 30°C for 6 hours and centrifuge at 5000rpm for 15 minutes to collect the bacteria.

[0088] 2. Biotransformation reaction

[0089] After centrifugation, the cells were added to the transformation medium, and t...

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Abstract

A method of producing ectoine by high-density culture of recombinant escherichia coli is provided. The method includes subjecting sodium L-aspartate to a biotransformation reaction by utilizing escherichia coli BW-p-BAD-ectABC having an accession number of CGMCC NO.8334. According to the method, 87.5 g of extracellular ectoine can be synthesized in each liter of fermenting bacteria after the fermenting bacteria are used repeatedly for five times, the synthetic efficiency is 11.67 g / L.d, and both the synthetic yield and the synthetic efficiency are higher than the reported synthesis levels. The method has great significance for industrial production and large-scale application of the ectoine.

Description

technical field [0001] The invention belongs to the field of microbial fermentation engineering, and in particular relates to a method for producing ectoine by microbial fermentation. Background technique [0002] Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid; ectoine) is the most widely used osmotic regulator in salt-tolerant and halophilic microorganisms. The ectoine molecule is highly water-soluble and uncharged. In a high-salt environment, cells can increase the intracellular osmotic pressure through the high concentration accumulation of ectoine, but it will not affect the normal physiological functions of intracellular biomacromolecules. Studies have shown that ectoine can provide protection to nucleic acids, proteins, cell membranes, and entire cells under stressful conditions such as high salt, high temperature, freezing, and drying. Therefore, it has broad application prospects in the fields of biological agents, cosmetics production, and pharmace...

Claims

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Application Information

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IPC IPC(8): C12P17/12C12R1/19
Inventor 董志扬何永志张山毕建成
Owner 南京众惠生物材料科技有限公司
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