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Rosev analogs and genetically engineered bacteria producing the rosavi analogs and applications thereof

A technology of genetically engineered bacteria and analogs, applied in the field of genetic engineering, can solve the problems of single species and low yield of rosavidin compounds, and achieve the effect of important scientific research value and social benefit.

Active Publication Date: 2022-03-08
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

[0004] The current types of rosavirin compounds are relatively single, and the yield is low

Method used

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  • Rosev analogs and genetically engineered bacteria producing the rosavi analogs and applications thereof
  • Rosev analogs and genetically engineered bacteria producing the rosavi analogs and applications thereof
  • Rosev analogs and genetically engineered bacteria producing the rosavi analogs and applications thereof

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Embodiment approach

[0041] According to a specific embodiment of the present invention, the exogenous gene of the genetically engineered bacteria is a gene capable of encoding tyrosine ammonia lyase TAL, a gene capable of encoding 4-coumaric acid-CoA ligase 4CL, and a gene capable of encoding cinnamon The gene of acyl-CoA reductase CCR and the gene capable of encoding UDP glucosyltransferase UGT can biosynthesize the new compound Triandrin B shown in the above formula IV, and can also biosynthesize coumaryl alcohol and coumaryl alcohol hydroxy monoglucoside Triandrin.

[0042] According to a specific embodiment of the present invention, the exogenous gene of the genetically engineered bacteria is a gene capable of encoding tyrosine ammonia lyase TAL, a gene capable of encoding 4-coumaric acid-CoA ligase 4CL, and a gene capable of encoding cinnamon The gene of acyl-CoA reductase CCR, the gene that can encode UDP glucosyltransferase UGT and the gene that can encode the glycosyltransferase that can ...

Embodiment 1

[0093] This example is used to illustrate the remodeling of the synthetic pathway of rosevin B in Escherichia coli

[0094] Escherichia coli expression vector V1 is pCDFDuet-4CL-PAL-CCR, the preparation method of this vector refers to the patent application number 201611179577.9 (Liu Tao, Zhou Wei, Bi Huiping, Zhuang Yibin, Yin Hua, Ma Yanhe. Recombinant Escherichia coli producing cinnamyl alcohol and redox , construction methods and applications).

[0095] Escherichia coli expression vector V2 is pRSFDuet-AtUGT73C5-CaUGT3, the preparation method of this vector is as follows:

[0096] (1) With the primer 73C5-5FPBam (SEQ ID No.5) / 73C5-3RPSal (SEQ ID No.6) as the guide, and the synthetic AtUGT73C5 gene (SEQ ID No.3) as the template, perform PCR to amplify AtUGT73C5 The gene was digested with BamHI and SalI and connected into the plasmid pRSDet1 digested with BamHI and SalI to construct pRSDDuet-AtUGT73C5.

[0097] (2) 5'-KpnI and 3'-XhoI restriction sites were added to both e...

Embodiment 2

[0103] This example is used to illustrate the remodeling of the rosavirin B synthesis pathway in high-phenylalanine-producing Escherichia coli

[0104] High-production phenylalanine Escherichia coli does not express tyrR (DNA-binding transcriptional dualregulator TyrR), tyrA (fused chorismate mutase / prephenate dehydrogenase), trpE (anthranilate synthase subunit TrpE) genes, and the specific construction method is as follows:

[0105] (1) Primer tyrR-5FPL (SEQ ID No: 7) / tyrR-3RPL (SEQ ID No: 8) is used to amplify Kan gene in pKD4 to replace tyrR gene coding region; Primer tyrRGD-5FP (SEQ ID No: 9 ) / tyrRGD-3RP (SEQ ID No: 10) is a primer for identifying recombinant bacteria after tyrR gene knockout, and the two primers are respectively located on the left and right sides of the target sequence region. The PCR reaction system is: 5×PCR buffer, 10 μL; dNTP (2mM each), 5 μL; primers tyrR-5FPL / tyrR-3RPL (10 μM), each 2 μL; Phusion high-fidelity DNA polymerase (5U / μL), 0.5 μL; pKD4,...

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Abstract

The present invention relates to the field of genetic engineering, in particular to a rosavir analog, a genetically engineered bacterium producing the rosavir analog, and an application thereof. The rosevin analog has a structure shown in formula I, formula II, formula III or formula IV, and the genetically engineered bacterium contains phenylalanine ammonia lyase or tyrosine ammonia lyase, 4-coumaric acid coenzyme A Genes for ligase 4CL, cinnamoyl-CoA reductase CCR, and UDP glucosyltransferase UGT. The genetically engineered bacteria can ferment and produce Triandrin and Triandrin B (Formula IV); or Triandrin, Triandrin B, Triandrin C (Formula II) and Triandrin D (Formula III), and the yield of Triandrin and Triandrin B can reach up to 1.7g / L and 1.6g / L; or Luosaiwei B (formula I), the highest output can reach 1.6g / L.

Description

technical field [0001] The present invention relates to the field of genetic engineering, in particular to a rosavir analog, a genetically engineered bacterium for producing the rosavir analog, an application of the genetically engineered bacterium in the production of a rosevin analog, and a method for producing A method for closev analogues. Background technique [0002] Cinnamyl alcohol glycosides (CAGs) are an important active ingredient in the endangered plant Rhodiola rosea, which has the functions of anti-oxidation, heart protection, and enhancement of learning and memory. Such compounds include roserin, roserin, roserin, roserin xyloside, Triandrin and the like. Rosev, rosevi and roserin are collectively referred to as rosavirin complex, in which rosavir is the main active ingredient and an important marker commonly used to evaluate the quality of Rhodiola rosea extract. Studies have found that the rosevin complex has a variety of beneficial pharmacological effects...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12R1/19
CPCC07H15/18C07H15/203C12N9/88C12Y403/01024C12N9/93C12N9/0008C12Y602/01012C12Y102/01044C12Y403/01023C12N9/1048C12N15/70C12P19/44C12P19/18
Inventor 刘涛毕慧萍王帅庄以彬马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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