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Sugar chain extension glycosyl transferase mutant, coding gene thereof, genetically engineered bacterium and application of mutant, coding gene and genetically engineered bacterium

A technology of glycosyltransferase and genetically engineered bacteria, applied in the field of genetic engineering, can solve the problems of many chemical synthesis steps, serious environmental pollution, and high cost, and achieve important scientific research value and social benefits

Active Publication Date: 2022-02-11
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are many chemical synthesis steps, high yield and low cost, and serious environmental pollution.

Method used

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  • Sugar chain extension glycosyl transferase mutant, coding gene thereof, genetically engineered bacterium and application of mutant, coding gene and genetically engineered bacterium
  • Sugar chain extension glycosyl transferase mutant, coding gene thereof, genetically engineered bacterium and application of mutant, coding gene and genetically engineered bacterium
  • Sugar chain extension glycosyl transferase mutant, coding gene thereof, genetically engineered bacterium and application of mutant, coding gene and genetically engineered bacterium

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Experimental program
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Effect test

Embodiment approach

[0046] According to a preferred embodiment of the present invention, the gene encoding phenylalanine ammonia lyase PAL is the AtPAL gene from Arabidopsis thaliana, GenBank: AY133595.1.

[0047] According to a preferred embodiment of the present invention, the gene encoding 4-coumaric acid-CoA ligase 4CL is shown in SEQ ID No.5, or the Pc4CL gene from parsley, GenBank: X13325.1, preferably as SEQ ID Shown in No.5.

[0048] According to a preferred embodiment of the present invention, the gene encoding cinnamoyl-CoA reductase CCR is the AtCCR gene from Arabidopsis thaliana, GenBank: AF332459.1.

[0049] According to a preferred embodiment of the present invention, the gene encoding UDP glucosyltransferase UGT73C5 is as shown in SEQ ID No.4, or is the AtUGT73C5 gene from Arabidopsis thaliana, GenBank: KJ138865.1, preferably as shown in SEQ ID No.4 Show.

[0050] According to a preferred embodiment of the present invention, the gene encoding UDP-glucose dehydrogenase is the endo...

Embodiment 1

[0084] This example is used to illustrate the acquisition of sugar chain elongation glycosyltransferase mutant CaUGT3-TN

[0085] Using the synthetic CaUGT3 gene (SEQ ID No.1) as a template, primers CaUGT3-5FPNde (SEQ ID No.7) and primers CaUGT3-3RPBam (SEQ ID No.8) were used for PCR amplification, and the amplified products were subjected to NdeI and BamHI After enzyme digestion, it was connected into the plasmid pET28a digested with NdeI and BamHI, and then transformed into Escherichia coli DH5α. After amplification, the plasmid was extracted, and the sequence of CaUGT3 was verified to be correct by sequencing, and pET28a-CaUGT3 was constructed.

[0086] By Tang method (Tang, L., Gao, H., Zhu, X., Wang, X., Zhou, M., Jiang, R., 2012. Construction of “small-intelligent” focused mutagenesis libraries using well-designed combinatorial degenerate primers.BioTechniques 52,149-158.), designed four mutation primers to introduce twenty natural amino acids into the mutation library u...

Embodiment 2

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

[0104] High-production phenylalanine Escherichia coli strain BPHE does not express tyrR (DNA-binding transcriptional dual regulator TyrR), tyrA (fused chorismate mutase / prephenate dehydrogenase), trpE (anthranilate synthase subunit TrpE) genes, and its construction method refers to patent application number 201910403327.6 (Liu Tao , Bi Huiping, Wang Shuai, Zhuang Yibin, Ma Yanhe. Rosev analogs and genetically engineered bacteria producing the rosavi analogs and their applications).

[0105] 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).

[0106] Escherichi...

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Abstract

The invention relates to the field of gene engineering, in particular to a sugar chain extension glycosyl transferase mutant, a coding gene thereof, a genetically engineered bacterium and application of the mutant and the coding gene and the genetically engineered bacterium. The genetically engineered bacterium contains a gene for coding phenylalnine ammonialyase PAL, a gene for coding 4-coumaric acid coenzyme A ligase 4CL, a gene for coding cinnamyl coenzyme A reductase CCR, a gene for coding UDP glucosyltransferase UGT, a gene for coding UDP glucose dehydrogenase, a gene for coding UDP xylose synthase and a gene and a gene CaUGT3-TN of a sugar chain extension glycosyltransferase mutant. The genetically engineered bacterium can be used for fermentation production of rosavin E (formula I), and the highest yield can reach 0.47 g / L.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a sugar chain elongation glycosyltransferase mutant and its coding gene, genetically engineered bacteria and their application in preparing rosavirin. Background technique [0002] Luosai, Luosaiwei and Luosailin are collectively referred to as Luosaiwei compound, which is an important active ingredient in the endangered plant Rhodiola rosea, which has the functions of anti-oxidation, heart protection, and enhancing learning and memory. Among them, rosavir is the main active ingredient, and it is also 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, such as intelligence development, anti-cancer, immune enhancement, anti-depression, and anti-ultraviolet radiation. [0003] Cinnamyl-(6'-O-β-D-xylopyranosyl)-O-β-D-glucopyranosi de, 2003 for the fi...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N1/21C12P19/44C12R1/19
CPCC12N9/1048C12N9/88C12N9/93C12N9/0008C12N9/0006C12N9/1051C12N15/52C12P19/44C12Y403/01024C12Y602/01012C12Y102/01044C12Y101/01022C12Y401/01035
Inventor 刘涛毕慧萍孙周通曲戈王帅庄以彬马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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