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Glycosyl transferase for catalyzing synthesis of gastrodin or salidroside, gene coding glycosyl transferase and application

A technology of glycosyltransferase and salidroside, which is applied in the direction of transferase, application, genetic engineering, etc., can solve the difficult operation and control of salidroside process, the low efficiency of gastrodin and salidroside, and the Problems such as long incubation period

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

[0015] The process of chemical synthesis of gastrodin 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 process of chemical synthesis of salidroside is long and difficult Operational control, high cost, difficult to achieve industrialization; microbial transformation of gastrodin and salidroside is inefficient, and exogenous substrates need to be added; plant tissue culture has a long reaction cycle and low titer
So far, there is no report on the de novo synthesis of gastrodin and salidroside by microorganisms

Method used

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  • Glycosyl transferase for catalyzing synthesis of gastrodin or salidroside, gene coding glycosyl transferase and application
  • Glycosyl transferase for catalyzing synthesis of gastrodin or salidroside, gene coding glycosyl transferase and application
  • Glycosyl transferase for catalyzing synthesis of gastrodin or salidroside, gene coding glycosyl transferase and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] gene ugt73b6 MK and Escherichia coli expression vector pET28a-ugt73b6 MK Get(method(method:

[0045] Random mutation of ugt73b6 (SEQ ID No: 3, which has been reported in the literature, derived from Rhodiola sachalinensis) was carried out by error-PCR, and 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 MK Escherichia coli expression vector pET28a-ugt73b6 MK . The 264th methionine of the specific glycosyltransferase ugt73b6 is mutat...

Embodiment 2

[0049] Construction of Escherichia coli containing the gene of the glycosyltransferase that catalyzes the synthesis of gastrodin or salidroside shown in SEQ ID No: 2

[0050] Plasmid pET28a-ugt73b6 MK Transformed into E. coli strain BL21(DE3) by chemical transformation:

[0051] Take 100 μL of competent E. coli strain BL21(DE3) cells on ice, add 2 μL of plasmid pET28a-ugt73b6 after 10 minutes MK , mix gently, place on ice for 30 minutes, heat shock at 42°C for 90 seconds, take it out and put it on ice for 2 minutes, add 600 μL 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 MK ), and by extracting the plasmid for enzyme digestion verification, the Escherichia coli strain BL21(DE3, pET28a-ugt73b6 MK ).

Embodiment 3

[0053] Strain BL21(DE3, pET28a-ugt73b6 MK ) fermentation culture

[0054] Strain BL21(DE3, pET28a-ugt73b6 MK ) 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.

[0055] Then the seed solution was transferred into 50 mL of M9Y (M9 basic medium+0.025% yeast extract) liquid with 50 mg / L kanamycin and 2 mM p-hydroxybenzyl alcohol according to the transfer amount (0.5 mL) of 1 volume % Culture 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 MK ) fermentation broth.

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Abstract

The invention discloses glycosyl transferase for catalyzing synthesis of gastrodin or salidroside, a gene coding the glycosyl transferase and application of glycosyl transferase. The glycosyl transferase for catalyzing synthesis of gastrodin or salidroside is as shown in SEQ ID No.1. Experiments prove that the glycosyl transferase for catalyzing synthesis of gastrodin or salidroside can be used for respectively catalytically converting hydroxy-benzyl alcohol into gastrodin and converting tyrosol into salidroside, and the yield of gastrodin and salidroside can be remarkably 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 salidroside 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...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N1/21C12P19/44C12R1/19
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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