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Sugar chain elongation glycosyltransferase mutant and its coding gene as well as genetically engineered bacteria and their application

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

Active Publication Date: 2022-06-28
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

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

Method used

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  • Sugar chain elongation glycosyltransferase mutant and its coding gene as well as genetically engineered bacteria and their application
  • Sugar chain elongation glycosyltransferase mutant and its coding gene as well as genetically engineered bacteria and their application
  • Sugar chain elongation glycosyltransferase mutant and its coding gene as well as genetically engineered bacteria and their application

Examples

Experimental program
Comparison scheme
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Embodiment approach

[0047] According to a preferred embodiment of the present invention, the gene encoding phenylalanine ammonia lyase PAL is from Arabidopsis thaliana AtPAL Gene, GenBank: AY133595.1.

[0048] According to a preferred embodiment of the present invention, the gene encoding 4-coumaric acid coenzyme A ligase 4CL is shown in SEQ ID No. 5, or the gene from parsley Pc4CL Gene, GenBank: X13325.1, preferably as shown in SEQ ID No.5.

[0049] According to a preferred embodiment of the present invention, the gene encoding cinnamoyl-CoA reductase CCR is from Arabidopsis thaliana AtCCR Gene, GenBank: AF332459.1.

[0050] According to a preferred embodiment of the present invention, the gene encoding UDP glucosyltransferase UGT73C5 is shown in SEQID No. 4, or the gene from Arabidopsis thaliana AtUGT73C5 Gene, GenBank: KJ138865.1, preferably as shown in SEQ ID No.4.

[0051] According to a preferred embodiment of the present invention, the gene encoding UDP-glucose dehydrogenase is end...

Embodiment 1

[0085] This example is used to illustrate the acquisition of the sugar chain extension glycosyltransferase mutant CaUGT3-TN

[0086] synthetic CaUGT3 The gene (SEQ ID No. 1) was used as the template, and the primers CaUGT3-5FPNde (SEQ ID No. 7) and CaUGT3-3RPBam (SEQ ID No. 8) were used for PCR amplification. Nde I and Bam After HI digestion, the Nde I and Bam The plasmid pET28a digested by HI was transferred into E. coli DH5α, and the plasmid was extracted after amplification. The sequence of CaUGT3 was verified by sequencing, and pET28a-CaUGT3 was constructed.

[0087] Through the Tang method (Tang, L., Gao, H., Zhu, X., Wang, X., Zhou, M., Jiang, R., 2012. Construction of “small-intelligent” focused mutagenesislibraries using well-designed combinatorial degenerate primers. BioTechniques52, 149-158.), designed four mutation primers to introduce 20 natural amino acids into the mutation library uniformly and without redundancy, and constructed a saturated mutant libra...

Embodiment 2

[0104] This example is used to illustrate the remodeling of the synthetic pathway of rosavide E in high-producing phenylalanine Escherichia coli

[0105] High phenylalanine-producing Escherichia coli strain BPHE does not express tyrR (DNA-binding transcriptionaldual regulator TyrR), tyrA (fused chorismate mutase / prephenate dehydrogenase) ， trpE (anthranilate synthase subunit TrpE) gene, and its construction method refers to patent application number 201910403327.6 (Liu Tao, Bi Huiping, Wang Shuai, Zhuang Yibin, Ma Yanhe. Loxavir analogs and genetically engineered bacteria producing the Loxavir analogs and applications thereof. ).

[0106] Escherichia coli expression vector V1 is pCDFDuet-4CL-PAL-CCR. The preparation method of this vector refers to patent application number 201611179577.9 (Liu Tao, Zhou Wei, Bi Huiping, Zhuang Yibin, Yin Hua, Ma Yanhe. Recombinant Escherichia coli for the production of cinnamyl alcohol and Luosai , construction methods and applications)....

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Abstract

The invention relates to the field of genetic engineering, in particular to a sugar chain extension glycosyltransferase mutant and its coding gene, genetically engineered bacteria and their application. The genetically engineered bacterium contains a gene encoding phenylalanine ammonia lyase PAL, a gene encoding 4-coumaric acid-CoA ligase 4CL, a gene encoding cinnamoyl-CoA reductase CCR, and a gene encoding UDP glucosyltransferase UGT. Gene, gene encoding UDP glucose dehydrogenase, gene encoding UDP xylose synthase and gene and sugar chain elongation glycosyltransferase mutant gene CaUGT3‑TN. The genetically engineered bacterium can ferment and produce rosevin E (formula I), and the maximum yield can reach 0.47g / L.

Description

technical field [0001] The present invention relates to the field of genetic engineering, in particular, to a sugar chain extension glycosyltransferase mutant and its encoding gene, as well as genetically engineered bacteria and their application in the preparation of rosavide E. Background technique [0002] Luosai, Luosaiwei and Luoceline are collectively referred to as Luosaiwei complex, which is an important active ingredient in the endangered plant Rhodiola rosea. It has the functions of anti-oxidation, protecting the heart, enhancing learning and memory. Among them, Luosaiwei is the main active ingredient, and it is also an important marker often used to evaluate the quality of Rhodiola rosea extract. Studies have found that Loxavir complex has a variety of beneficial pharmacological effects, such as nootropics, anti-cancer, enhancing immunity, anti-depression, anti-ultraviolet radiation and so on. [0003] Cinnamyl-(6'- O - β -D-xylopyranosyl)- O-β -D-glucopyran...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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