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A kind of recombinant Escherichia coli producing salidroside and its construction method and application

A technology for recombining Escherichia coli and Escherichia coli, which is applied in the field of bioengineering, can solve the problem of low yield and achieve the effect of increasing yield

Active Publication Date: 2020-03-31
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

[0011] The above invention has achieved de novo synthesis of salidroside in Escherichia coli, but the yield is low. Therefore, increasing the yield of salidroside has important scientific research value and social benefits

Method used

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  • A kind of recombinant Escherichia coli producing salidroside and its construction method and application
  • A kind of recombinant Escherichia coli producing salidroside and its construction method and application
  • A kind of recombinant Escherichia coli producing salidroside and its construction method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] gene UGT73B6 MK and Escherichia coli expression vector pET28a‐UGT73B6 MK get

[0042] Referring to the Chinese patent (patent application number 201510160496.3) that catalyzes the glycosyltransferase that synthesizes gastrodin or salidroside and the gene encoding the enzyme and its application, the sequence of UGT73B6 (SEQ ID No: 1, which has been reported in the literature, is derived from plants Rhodiola sachalinensis) were subjected to error‐PCR random mutation, and the obtained fragment was digested and ligated into the commercial vector pET28a, and then screened by directed evolution (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 novobio...

Embodiment 2

[0045] Escherichia coli expression vector pETDuet‐ARO10‐UGT73B6 MK &pBbA5c-T7Pol-lac pgm-lac galU, the preparation method of the carrier preferably comprises the following steps:

[0046] (a) With reference to Chinese patent (patent application number: 201410115011.4.), Bi Huiping, Bai Yanfen, etc., a high-yielding E. coli expression strain of tyrosol and / or salidroside and icariside D2 and its application, with primer aro ‐5FPNco (SEQ ID No: 3) / aro‐3RPSac (SEQ ID No: 4) was used as a guide, and the genomic DNA of Saccharomyces cerevisiae (Saccharomycescerevisiae) S288C was used as a template for PCR to amplify the ORF of ARO10, and the amplified product was passed through NcoI After digestion with NcoI and SacI, it was connected into the plasmid pETDuet-1 digested with NcoI and SacI, and the plasmid pETDuet-ARO10 was constructed. As well as the Chinese patent (patent application number 201510160496.3) catalyzing the glycosyltransferase for the synthesis of gastrodin or salid...

Embodiment 3

[0049] This embodiment is used to illustrate the construction of Escherichia coli bacterial strain BMGU

[0050](1) According to the present invention, the method for making feaB, tyrR, pykA, pykF, pheA, galE, galT and ugd genes not expressed on the E. coli chromosome can be a conventional method in the art, for example, by gene knockout It can also be done by gene silencing methods. The present invention is preferably carried out by gene knockout method. In the present invention, there is no special requirement for the gene knockout method, and various methods capable of gene knockout in Escherichia coli can be used, such as using the λRed gene knockout system. With reference to the Chinese patent (patent application number: 201410115011.4.), Bi Huiping, Bai Yanfen, etc., a high-yielding E. coli expression strain of tyrosol and / or salidroside and icariside D2 and its application, with E. coli △ A as the Starting strain, plasmid pKD46 (temperature-sensitive, containing exo, ...

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Abstract

The invention discloses recombinant Escherichia coli producing salidroside, a construction method and applications thereof. The construction method comprises: (1) using Escherichia coli [delta]A as a starting strain, and carrying out gene knockdown or gene silencing to make the galE gene, the galT gene and the ugd gene on the Escherichia coli chromosome be not expressed to obtain an Escherichia coli strain BMGU; and (2) introducing genes such as ARO10, UGT73B6MK, pgm, galU and T7Polymerase to over-express the genes such as ARO10, UGT73B6MK, pgm, galU and T7Polymerase so as to achieve the heterologous synthesis of salidroside, such that the recombinant Escherichia coli producing salidroside is obtained. According to the present invention, the efficient UDP-glucosyltransferase mutant UGT73B6MK is introduced, and the glucose metabolism pathway is optimized, such that the yield of salidroside is significantly improved.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to a recombinant Escherichia coli for producing salidroside, a construction method and application thereof. Background technique [0002] Rhodiola rosea is a rare wild plant that grows in the alpine and pollution-free areas. It is a traditional medicine of the Tibetan people in my country. It has been used for more than 1,000 years. It has the functions of stimulating the nervous system, increasing work efficiency, eliminating fatigue and preventing altitude sickness. In addition, Rhodiola also has the functions of protecting cardiovascular and cerebrovascular, nerve cells, anti-tumor and anti-radiation. The main medicinal active ingredient of Rhodiola rosea is salidroside and its aglycon tyrosol. In recent years, there are more and more products such as pharmaceuticals, beverages, food and cosmetics produced with salidroside as the main raw material. Its aglycon ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12N1/21C12P19/44C12R1/19
CPCC07K14/245C12N9/1048C12N9/1247C12N9/88C12N15/70C12N2800/101C12P19/44C12Y207/07006C12Y401/0108
Inventor 刘涛毕慧萍庄以彬殷华孙雪马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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