Recombinant bacteria for producing salidroside and analogue thereof, and construction method and use

A technology of salidroside and its analogues, which is applied in the field of recombinant bacteria producing salidroside and its analogues and construction, can solve problems such as unfavorable industrial production, achieve industrial production, solve source problems, and reduce production cost effect

Inactive Publication Date: 2017-12-12
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the chemical synthesis of salidroside and its analogues has become increasingly mature, most of them require selective protection, activation or the use of expensive metal catalysts
Therefore, the above methods are not conducive to industrial production

Method used

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  • Recombinant bacteria for producing salidroside and analogue thereof, and construction method and use
  • Recombinant bacteria for producing salidroside and analogue thereof, and construction method and use
  • Recombinant bacteria for producing salidroside and analogue thereof, and construction method and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Design of ketone decarboxylase gene synkdc and glycosyltransferase gene synyjic

[0043] Preferably, the Pichia pastoris GS115 keto-decarboxylase gene and the Bacillus licheniformis glycosyltransferase gene, the amino acid sequences of which are respectively shown in SEQ ID No.01 and SEQ ID No.02 in the sequence listing. Use the JCAT online codon optimization software (http: / / www.jcat.de) in combination with the OPTIMIZER online codon optimization tool (http: / / genomes.urv.es / OPTIMIZER / ) to perform codon preference in Escherichia coli Optimizing and designing the full-length synkdc gene and synyjic gene, as shown in the sequence table SEQ ID No.03 and SEQ ID No.04 respectively.

Embodiment 2

[0044] Example 2 λ-red homologous recombination method T7 RNA polymerase was integrated into the chromosome of the chassis strain SyBE-002447.

[0045] The detailed steps of the construction process of the chassis strain integrating T7RNA polymerase into the chromosome of the chassis strain SyBE-002447 are as follows:

[0046] 1. Design the primers T7-RNA F and T7-RNA R sequences respectively as shown in the sequence table SEQ ID NO.07, SEQ ID NO.08 to obtain the T7 RNA polymerase gene from Escherichia coli BL21 (DE3) bacteria, and the T7 RNA polymerase The gene sequence is as shown in the sequence listing SEQ ID NO.05.

[0047] 2. The sequences of primers T7F and T7R, T7-Chl F and T7-Chl R are as shown in the sequence table SEQ ID NO.09, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, prepared by overlap extension PCR with chlorine Mycin-resistant T7 RNA polymerase fragment.

[0048] 3. Introduce the pKD46 plasmid into the strain SyBE-002447 to obtain SyBE-002447 / pKD46, inoculate...

Embodiment 3

[0051] Example 3 Construction of recombinant expression vector pSynkdc1-yjic

[0052] The synkdc gene fragment obtained by chemical total synthesis was added to the two ends of the fragment by PCR method, respectively, and the EcoRI and PstI restriction sites located downstream of the first promoter of the expression vector pRSFDuet-1 were added, and the FastDigest endonuclease EcoRI was used to Digest with PstI, the reaction system is: 5 μL 10*FD buffer, 2.5 μL EcoRI, 2.5 μL PstI, 30 μL synkdc gene fragment and 10 μL ultrapure water. The reaction conditions are: 37°C, 2h. Using a PCR purification kit, add 250 μL of Bingding Buffer solution to 50 μL of the digested product, mix well, add to the adsorption column, let stand for one minute, centrifuge at 10,000 g for 1 minute, and discard the effluent. Add 650μL Wash Buffer, centrifuge at 10,000g for 1 minute, and discard the effluent. Centrifuge at 10,000g for 2 minutes to remove residual Wash Buffer. Put the adsorption colu...

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Abstract

The invention discloses recombinant bacteria for producing salidroside and analogue thereof, and a construction method and use. The construction method comprises the steps of synthesizing keto-decarboxylase gene synkdc and glycosyl transferase gene synyjic, and connecting to a vector pRSFDuet-1 to construct a recombinant vector pSynkdc1-yjic; enabling T7RNA polymerase gene to be free or integrated to SyBE-002447 base bacterial strain, and introducing pSynkdc1-yjic to obtain SyBE-218011 and SyBE-218013 bacterial stains; and directly integrating synkdc and synyjic to SyBE-002447 chromosome, and removing feaB and ushA in a knockout manner to obtain a SyBE-218015 bacterial strain. The bacterial strain disclosed in the invention can be used for producing salidroside and analogue thereof through fermentation, so that the problem existing in source of the salidroside and analogue thereof can be solved, and cost can be lowered.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to a recombinant bacterium producing salidroside and its analogs and a construction method. Background technique [0002] Salidroside (salidroside) is the main active ingredient of Rhodiola rosea (Crassulaceae) Rhodiola plant, which has good anti-oxidation, anti-radiation, anti-fatigue, immune regulation, and anti-altitude hypoxia. and other pharmacological activities. Its molecular formula is C 14 h 20 o 7 , molecular weight 300.30, off-white or light yellow powder. In recent years, studies have found that salidroside can be used in the treatment of cancer, nervous system diseases, cell aging, hypoxia and ischemia in brain and heart, cognitive impairment, skin spots, metabolic regulation, etc. Sedroside is widely used in industrial production such as pharmaceuticals, health care products, and cosmetics. [0003] So far, the main source of salidroside is still wild rhodiola....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N1/21C12P19/44C12R1/19
CPCC12N9/1048C12N9/88C12N15/70C12N2800/101C12P19/44
Inventor 赵广荣李晓波刘雪
Owner TIANJIN UNIV
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