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CN-121991914-A - Glycosyltransferase gene for catalyzing astragalin synthesis and application thereof

CN121991914ACN 121991914 ACN121991914 ACN 121991914ACN-121991914-A

Abstract

The invention provides a glycosyltransferase gene for catalyzing astragalin synthesis and application thereof, and belongs to the technical field of biology. The invention develops a new glycosyltransferase, a new glycosyltransferase mutant is obtained by utilizing a genetic engineering method, and astragalin can be synthesized by fermenting microorganisms or in-vitro enzyme reaction processes. The method has the advantages of simpler synthesis path, mild synthesis condition, less environmental pollution, higher synthesis efficiency, better purity and less waste of environmental resources, provides a new synthesis path for synthesizing the chemical substance of astragalin with high economic value, and provides a new thought and method for actual production.

Inventors

  • REN CHAOXIANG
  • LI YUHANG
  • PEI JIN
  • CHEN JIANG
  • Xi Ziqing
  • XIAN BIN
  • CHEN CHAO

Assignees

  • 成都中医药大学

Dates

Publication Date
20260508
Application Date
20241106

Claims (10)

  1. 1. A mutant glycosyltransferase, which is obtained by mutation based on glycosyltransferase, wherein the amino acid sequence of glycosyltransferase is shown in SEQ ID No.1, and the mutation comprises at least one of the following sites: (1) Mutating 276 th amino acid from S to T; (2) Mutation of amino acid 384 from E to D; (3) Mutating the 15 th amino acid from G to T; (4) Mutation of amino acid 136 from T to P; (5) The 137 th amino acid is mutated from S to G.
  2. 2. The mutant glycosyltransferase of claim 1, wherein the amino acid sequence of the mutant glycosyltransferase is shown in SEQ ID No. 3.
  3. 3. A gene encoding the mutant glycosyltransferase of claim 1, wherein the nucleotide sequence is set forth in SEQ ID No. 4.
  4. 4. A recombinant expression plasmid comprising the gene according to claim 3.
  5. 5. A recombinant genetically engineered bacterium, which comprises the recombinant expression plasmid of claim 4.
  6. 6. Use of the gene of claim 3, the recombinant expression plasmid of claim 4 or the recombinant genetically engineered bacterium of claim 5 for the preparation of the mutant glycosyltransferase of claim 1.
  7. 7. A method of making the mutant glycosyltransferase of claim 1, comprising the steps of: (1) Constructing the recombinant expression plasmid of claim 4; (2) And (3) introducing the recombinant expression plasmid into a host cell, and inducing expression to obtain the mutant glycosyltransferase.
  8. 8. The method of claim 7, wherein in the step (1), an original expression plasmid containing a glycosyltransferase gene and a specific primer are subjected to PCR amplification to obtain a recombinant expression plasmid, wherein an amplification system of the PCR amplification comprises the specific primer of 276 th amino acid, 384 th amino acid, 15 th amino acid, 136 th amino acid and 137 th amino acid, the PCR amplification condition is 90-100 ℃, 2-4 min, 90-100 ℃ 20-40 s, 70-75 ℃ 2-4 min, 30-40 cycles, and 70-75 ℃ 6-10 min, and a recombinant expression vector for constructing the recombinant expression plasmid comprises one or more than two of an escherichia coli expression vector, a yeast expression vector, a bacillus subtilis expression vector, a lactobacillus expression vector, a streptomycete expression vector, a phage vector, a filamentous fungus expression vector, a plant expression vector, an insect expression vector or a mammalian cell expression vector; in step (2), the host cell is an E.coli host cell, a yeast host cell, a Bacillus subtilis host cell, a Lactobacillus host cell, an actinomycete host cell, a filamentous fungus host cell, an insect cell, or a mammalian cell.
  9. 9. Use of the mutant glycosyltransferase of claim 1 for the synthesis of glycosylated flavonoid compounds.
  10. 10. The use according to claim 9, wherein the glycosylated flavonoid comprises kaempferol-3-O-glucoside, quercetin-3-O-glucoside, apigenin-3-O-glucoside, isorhamnetin-3-O-glucoside.

Description

Glycosyltransferase gene for catalyzing astragalin synthesis and application thereof Technical Field The invention belongs to the technical field of biology, and particularly relates to a glycosyltransferase gene for catalyzing astragalin synthesis and application thereof. Background Astragaloside is a natural maize color glycoside substance, which plays a key role in the accumulation and metabolism regulation process of the traditional Chinese medicine in safflower. Numerous studies have shown that astragalin can be synthesized in cells by using specific glycosyltransferases (UGTs). However, since astragalin has important medicinal value such as treatment of liver diseases, respiratory diseases, etc., research on the synthesis of astragalin has been paid attention to. In recent years, the research of the synthesis of astragalin is also rapid, and the main synthesis scheme is to extract and purify by a physicochemical method and then perform chemical synthesis. However, according to the related data research, the chemical synthesis method of astragalin has some disadvantages. First, current synthetic methods require the use of higher temperatures and pressures, which may lead to degradation or formation of some compounds. Second, the synthesis process requires the use of large amounts of organic solvents, which can be environmentally friendly. In addition, the synthesized compounds may require further purification and treatment to remove impurities and byproducts, which also require more resources and energy consumption. These disadvantages reflect the high cost and inefficiency of the synthetic method. Therefore, there is a need to develop efficient, environmentally friendly, economical synthetic methods. By using a genetic engineering method, a specific glycosyltransferase (UGT) gene is introduced into a microorganism, and astragalin and other important products are synthesized through metabolic pathways of the microorganism. Although this method has the advantages of stereoselectivity, green, environmental protection, economy, simplicity and convenience, etc. compared with the chemical synthesis method. However, the problems of low enzyme activity, poor stability, low conversion rate and the like are reported to exist when the method is applied to the biocatalysis preparation of the astragalin. Literature (DOI: 10.1021/acs.jafc.3c05117) reports that the glycosyltransferase function involved in the biosynthesis of safflower astragalin was identified in vivo using the safflower corolla protoplast system, and the catalytic mechanism of the enzyme was analyzed in combination with in vitro experiments, but the catalytic activity was not high. Disclosure of Invention In order to solve the problems in the prior art, the invention aims to provide a glycosyltransferase gene for catalyzing astragalin synthesis and application thereof. The invention provides a mutant glycosyltransferase, which is obtained by mutation on the basis of glycosyltransferase, wherein the amino acid sequence of the glycosyltransferase is shown as SEQ ID No.1, and the mutation comprises at least one of the following sites: (1) Mutating 276 th amino acid from S to T; (2) Mutation of amino acid 384 from E to D; (3) Mutating the 15 th amino acid from G to T; (4) Mutation of amino acid 136 from T to P; (5) The 137 th amino acid is mutated from S to G. Further, the amino acid sequence of the mutant glycosyltransferase is shown as SEQ ID No. 3. The invention also provides a gene for coding the mutant glycosyltransferase, and the nucleotide sequence of the gene is shown as SEQ ID No. 4. The invention also provides a recombinant expression plasmid which contains the gene. The invention also provides a recombinant genetically engineered bacterium which contains the recombinant expression plasmid. The invention also provides the application of the gene, the recombinant expression plasmid or the recombinant genetically engineered bacterium in preparing the mutant glycosyltransferase. The invention also provides a method for preparing the mutant glycosyltransferase, which comprises the following steps: (1) Constructing the recombinant expression plasmid; (2) And (3) introducing the recombinant expression plasmid into a host cell, and inducing expression to obtain the mutant glycosyltransferase. Further, in the step (1), carrying out PCR amplification on an original expression plasmid containing a glycosyltransferase gene and a specific primer to obtain a recombinant expression plasmid, wherein an amplification system of the PCR amplification comprises the specific primer of 276 th amino acid, 384 th amino acid, 15 th amino acid, 136 th amino acid and 137 th amino acid, the PCR amplification is carried out under the conditions of 90-100 ℃, 2-4 min, 90-100 ℃ 20-40 s, 70-75 ℃ 2-4 min, 30-40 cycles and 70-75 ℃ 6-10 min, and the recombinant expression vector for constructing the recombinant expression plasmid comprises one or more than two of an escherichi