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Glycosyl transferase synthesized by catalytic gastrodine as well as gene of encoding enzyme and application

A technology of glycosyltransferase and gastrodin, applied in the field of bioengineering, can solve the problems of low titer, low yield, low microbial conversion efficiency, etc., and achieve the effect of increasing yield

Active Publication Date: 2015-07-15
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The chemical synthesis process is relatively mature, but the addition of heavy metal catalysts will cause great damage to the environment; the chemical synthesis method does not require red phosphorus and bromine, and the yield is low; the conversion efficiency of microorganisms is low, and exogenous substrates need to be added ;Plant tissue culture reaction cycle is long, titer is low
So far, there is no report on the de novo synthesis of gastrodin by microorganisms

Method used

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  • Glycosyl transferase synthesized by catalytic gastrodine as well as gene of encoding enzyme and application
  • Glycosyl transferase synthesized by catalytic gastrodine as well as gene of encoding enzyme and application
  • Glycosyl transferase synthesized by catalytic gastrodine as well as gene of encoding enzyme and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] gene ugt73b6 FS 、ugt73b6 FS+MK and Escherichia coli expression vector pET28a-ugt73b6 FS , pET28a-ugt73b6 FS+MK How to get it:

[0043]ugt73b6 (SEQ ID No: 5, the sequence has been reported in the literature, derived from Rhodiola sachalinensis) was randomly mutated by error-PCR, the obtained fragment was digested and ligated into the commercial vector pET28a, and then directed Evolutionary screening (Richard W.Gantt, Pauline Peltier-Pain, Shanteri Singh, Maoquan Zhou, and Jon S. Thorson, Broadening the scope of glycosyltransferase-catalyzed sugar nucleotide synthesis, PNAS, 2013, 110(19): 7648-7653; Gavin J.Williams, Randal D.Goff, Changsheng Zhang, and Jon S.Thorson, Optimizing glycosyltransferase specificity via'hot spot'saturation mutagenesis presents a new catalyst for novobiocin glycorandomization, Chem Biol.2008,15(4):393-409) , get mutant gene ugt73b6 FS Escherichia coli expression vector pET28a-ugt73b6 FS . Specifically, the 389th phenylalanine of the glyc...

Embodiment 2

[0048] Construction of Escherichia coli containing the gene of the glycosyltransferase that catalyzes the synthesis of gastrodin shown in SEQ ID No: 3

[0049] Plasmid pET28a-ugt73b6 FS Transformed into E. coli strain BL21(DE3) by chemical transformation:

[0050] Take 100 μl of competent E. coli strain BL21(DE3) cells on ice, add 2 μl of plasmid pET28a-ugt73b after 10 minutes FS , mix gently, place on ice for 30 minutes, heat shock at 42°C for 90 seconds, take it out and place on ice for 2 minutes immediately, add 600μl LB liquid medium, revive and culture at 37°C, 150rpm shaker for 30 minutes, and then put the bacteria The solution was spread on LB plates containing kanamycin. Selection of Transformed Strain BL21(DE3,pET28a-ugt73b6) Carrying Expression Vector Using Kanamycin Resistance FS ), and by extracting the plasmid for enzyme digestion verification, the Escherichia coli strain BL21 (DE3, pET28a-ugt73b6 containing the gene of the glycosyltransferase that catalyzes ga...

Embodiment 3

[0055] (1) Strain BL21 (DE3, pET28a-ugt73b6 FS ) fermentation culture

[0056] Strain BL21(DE3, pET28a-ugt73b6 FS ) in 2 mL of LB liquid medium added with 50 mg / L kanamycin, and cultivated at 37° C. for 12 hours to obtain a seed liquid.

[0057] Then transfer the seed solution into 50ml M9Y (M9 basic medium+0.025%yeast extract) liquid culture with 50mg / L kanamycin and 2mM p-hydroxybenzyl alcohol according to the transfer amount (0.5ml) of 1 volume % medium, cultured at 37°C, when OD 600 At about 0.6, IPTG (isopropyl-β-D-thiogalactopyranoside) with a final concentration of 0.1 mM was added for induction, and culture was continued at 30° C. for 48 hours. The bacterial strain BL21(DE3, pET28a-ugt73b6 expressing gastrodin was obtained FS ) fermentation broth.

[0058] (2) Strain BL21 (DE3, pET28a-ugt73b6 FS+MK ) fermentation culture

[0059] With BL21 (DE3, pET28a-ugt73b6 FS+MK ) to replace the bacterial strain BL21 (DE3, pET28a-ugt73b6) in this example FS ), other with t...

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PUM

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Abstract

The invention discloses glycosyl transferase synthesized by catalytic gastrodine as well as a gene of encoding enzyme and application. The glycosyl transferase synthesized by catalytic gastrodine is shown in SEQ ID No:1; another glycosyl transferase synthesized by catalytic gastrodine is shown in SEQ ID No:2. According to the glycosyl transferase synthesized by two catalytic gastrodine, p-hydroxybenzyl alcohol can be catalyzed and converted into gastrodine, so that the yield of gastrodine can be obviously improved.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to two glycosyltransferases that catalyze the synthesis of gastrodin and their application. Background technique [0002] Gastrodia elata is the stem of the orchid plant Gastrodia elata B1., which is a commonly used and precious traditional Chinese medicine. The plant Gastrodia elata grows in sparse forests, in open spaces, forest edges, and shrub edges, at an altitude of 400-3200 meters. It has been rated as a vulnerable species by the International Union for Conservation of Nature (IUCN), and has been included in the "Endangered Species of Wild Animals and Plants". In Appendix II of the Convention on International Trade (CITES), it is also included in China's "List of National Key Protected Wild Plants (Second Batch)", and is a Class II protected plant. Its rhizome is used medicinally to treat dizziness, numbness of limbs, convulsions in children, epilepsy and c...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N1/21C12P17/06C12R1/19
CPCC12N9/1048C12P17/06
Inventor 刘涛白艳芬殷华毕慧萍庄以彬马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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