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Engineering bacterium for synthesizing salicin as well as construction method and application of engineering bacterium

A technology of salicin and engineering bacteria, applied in the field of bioengineering, can solve the problems of low extraction rate, low content of salicin, rare salicin and the like

Active Publication Date: 2021-06-25
BEIJING UNIV OF CHEM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Salicin from natural sources is mainly extracted from Salicaceae plants. For example, the invention patent with the Chinese application number CN2015109566550 and the name "A Method for Extracting Salicin from White Willow Bark" discloses a method of extracting salicin from white willow bark. The method for extracting salicin in the skin, the raw material white willow bark is extracted by percolation with weak acid-ethanol solution, which can improve the solubility of salicin in the alcohol solution. Although this method can improve the extraction rate of salicin, the salicin The content in Salixaceae plants is less, and the extraction rate is still low
And at present, there are few reports about the production of salicin by biotechnology

Method used

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  • Engineering bacterium for synthesizing salicin as well as construction method and application of engineering bacterium
  • Engineering bacterium for synthesizing salicin as well as construction method and application of engineering bacterium
  • Engineering bacterium for synthesizing salicin as well as construction method and application of engineering bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Recombinant plasmid pZE-OSSGT1

[0046] The recombinant plasmid pZE-OSSGT1 provided in this example is mainly obtained by linking the group encoding glucosyltransferase (OSSGT1) to the Escherichia coli expression vector pZE12-luc.

[0047] The method for constructing the above-mentioned recombinant plasmid provided in this example specifically includes the following steps. Screening for genes encoding glucosyltransferase (OSSGT1) derived from bacteria, fungi or protein engineering. After the target fragment is obtained by PCR amplification of the target gene encoding glucosyltransferase (OSSGT1), the target fragment and the vector are digested with an appropriate enzyme, and the digested fragment is recovered, and then inserted into the expression plasmid On pZE12-luc, obtain the pZE-OSSGT1 recombinant plasmid (see Table 1). Specifically, in this example, the glucosyltransferase (OSSGT1) is derived from Akita Komachi rice.

Embodiment 2

[0048] Example 2 Engineering bacteria for synthesizing salicin: recombinant Escherichia coli BW - an Escherichia coli strain containing the pZE-OSSGT1 recombinant plasmid

[0049] The engineering bacteria for synthesizing salicin provided by the present invention have no special requirements on the type of host strain used to construct the expression plasmid. In the embodiment of the present invention, strain BW25113 is used as the initial host for constructing the plasmid.

[0050] First, pick fresh BW25113 colonies and inoculate them into 4 mL of LB medium, incubate at 37°C for 8–12 h, then inoculate 1 mL into 100 mL of LB medium, and incubate at 37°C until OD 600 When the growth reaches 0.6, centrifuge at 6000 rpm for 10 min at 4 °C to collect the bacteria, wash with 10 mL of 10% pre-cooled glycerol, centrifuge at 6000 rpm for 10 min, repeat the glycerol washing step, and drain the remaining glycerol as much as possible after centrifugation. Finally, an appropriate amount o...

Embodiment 3

[0051] Example 3 Application of Recombinant Escherichia coli BW: Combining Salicyl Alcohol to Produce Salicin by Fermentation

[0052] The application of recombinant Escherichia coli BW in the production of salicin includes: picking fresh recombinant Escherichia coli BW engineering single colonies on the plate and inoculating them into 4 mL LB test tubes containing the corresponding antibiotics, culturing at 37°C for 12 h, and then transferring to the tubes containing the corresponding antibiotics. Fermentation was carried out in shake flasks with 50 mL of M9 medium, the inoculum size was 1% by volume, the fermentation temperature was 37°C, and the rotation speed was 220rpm; wherein, the M9 medium included: 2.5 g / L glucose, 20 g / L Glycerin, 5 g / L yeast powder, 6 g / L Na 2 HPO 4 , 0.5 g / L NaCl, 3 g / L KH 2 PO 4 , 1g / L NH 4 Cl, 2g / L MOPS, 246.5 mg / L MgSO 4 ·7H 2 O and 14.7 mg / LCaCl 2 2H 2 O, and add corresponding antibiotics according to the actual situation. The inducer ...

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Abstract

The invention provides an engineering bacterium suitable for salicin, which comprises a host and a recombinant plasmid vector, and the recombinant plasmid vector is connected with a gene for expressing and coding glucosyltransferase. The recombinant plasmid vector is also connected with a gene for coding carboxylic acid reductase and phosphoryl transferase, which is co-expressed with the gene for coding glucosyltransferase. The recombinant plasmid vector is also connected with genes for over-expressing and coding shikimic acid kinase, pyruvate kinase, transketolase, 3-deoxy-7-phosphoenanthate synthase, iso-chorismate pyruvate lyase and isochorismate synthase; and the host is also knocked out of genes for coding pykA and pykF, so that the yield of salicin is increased. The invention also provides a construction method and an application of the engineering bacterium for synthesizing salicin. By utilizing the engineering bacteria for synthesizing the salicin, the salicin is produced by using glucose and / or glycerol and other carbon sources, so that efficient biosynthesis of the salicin is realized.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to an engineering bacterium for synthesizing salicin and its construction method and application. Background technique [0002] Salicin, English name: salicin, English alias: salicoside, CAS number: 138-52-3, molecular weight 286.28, molecular formula C 13 h 18 o 7 , the structural formula is: . [0003] Salicin is a salicylol glycoside that was first isolated from willow in 1828. Studies have found that salicin in willow bark extract (WBE) has anti-inflammatory and analgesic effects, because it is gradually transported to the lower intestinal tract, hydrolyzed by intestinal bacteria into salicyl alcohol, and converted into salicylic acid after absorption . And it produces an antipyretic effect without causing stomach damage. Therefore, salicin therapy may be useful in patients who develop gastric lesions while receiving strong analgesics such as NASAIDs. Other studi...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P19/44C12R1/19
CPCC12N9/1051C12N9/0008C12N9/12C12N9/88C12N9/1205C12N9/1085C12N9/1022C12P19/44C12Y204/01C12Y102/99006C12Y402/03005C12Y207/01071C12Y207/0104C12Y202/01001C12Y205/01054
Inventor 申晓林袁其朋赵千婧
Owner BEIJING UNIV OF CHEM TECH
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