Flavone-O-methyltransferase, and application thereof in synthesis of wogonin, isowogonin and moslosooflavone

A technology of methyltransferase and flavonoids of thalassemia, which is applied in the field of biology

Active Publication Date: 2021-09-10
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

[0005] Methylated flavonoids such as wogonin, isowogonin, and scutellaria flavonoids have been extracted from Scutellaria barbata, a plant of the Labiatae family, but there is no 8-position methylated flavonoi

Method used

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  • Flavone-O-methyltransferase, and application thereof in synthesis of wogonin, isowogonin and moslosooflavone
  • Flavone-O-methyltransferase, and application thereof in synthesis of wogonin, isowogonin and moslosooflavone
  • Flavone-O-methyltransferase, and application thereof in synthesis of wogonin, isowogonin and moslosooflavone

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Obtaining of Encoding Nucleotide and Construction of Vector

[0052] According to the nucleotide sequence information provided by the present invention (SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, SEQ ID No.11, SEQ ID No.12, SEQ ID No.13, SEQ ID No .14), performing gene synthesis. Using the shuttle plasmid YCPlac33-PE as the carrier, the exogenous sequence was inserted between the restriction sites SalI and XbaI, and the recombinant vector was constructed using the Gibson assembly method ( figure 1 ). In addition, for SEQ ID No.14, the shuttle plasmid YCPlac22-PE was also used as a carrier, and the foreign sequence was inserted between the restriction site SalI and XbaI, and the recombinant vector was constructed using the Gibson assembly method ( figure 2 ).

Embodiment 2

[0053] Example 2 Construction of Wogonin-producing Saccharomyces cerevisiae Host Bacteria

[0054] 2.1 Production of wogonin using nor-wogonin as substrate

[0055] Utilize conventional lithium acetate transformation method to comprise the recombination of nucleotide sequence SEQ ID No.8 or SEQ ID No.9 or SEQ ID No.10 or SEQ ID No.11 or SEQ ID No.12 described in embodiment 1 The vector was transformed into Saccharomyces cerevisiae host strain W303-1B, and the clones that could grow on uracil-deficient complete (CM) medium were picked, and wogonin was fermented with 1mM norwogonin as the substrate .

[0056] 2.2 Production of wogonin with chrysin as substrate

[0057] The recombinant vector comprising the nucleotide sequence SEQ ID No.12 described in Example 1 was transformed into a Saccharomyces cerevisiae host containing flavone-8-hydroxylase (F8H) and P450 reductase (CPR) using a conventional lithium acetate transformation method Bacteria, pick the clones that can grow on...

Embodiment 3

[0058] Example 3 Construction of Saccharomyces cerevisiae host bacteria producing iso-wogonin

[0059] 3.1 Production of iso-wogonin using nor-wogonin as substrate

[0060] The recombinant vector comprising nucleotide sequence SEQ ID No.13 or SEQ ID No.14 described in Example 1 is transformed into Saccharomyces cerevisiae host bacterium W303-1B by conventional lithium acetate transformation method, and picking can be carried out at uracil The clones grown on the deficient CM medium were fermented with norwogonin at a concentration of 1 mM to produce isowogonin.

[0061] 3.2 Production of isowogonin with chrysin as substrate

[0062] The recombinant vector comprising the nucleotide sequence SEQ ID No.14 described in Example 1 was transformed into a Saccharomyces cerevisiae host containing flavone-8-hydroxylase (F8H) and P450 reductase (CPR) using a conventional lithium acetate transformation method Bacteria, pick the clones that can grow on tryptophan and uracil-deficient CM ...

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Abstract

The invention provides flavone-O-methyltransferase, which comprises one or more of flavone-8-methyltransferase with the amino acid sequences as shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4 and SEQ ID No.5, and flavone-7-methyltransferase with the amino acid sequences as shown in SEQ ID No.6 and SEQ ID No.7 The invention also provides a nucleotide sequence for coding the amino acid sequence, a recombinant vector containing the polynucleotide sequence for coding the above amino acid, a recombinant host cell and application. The flavone-O-methyltransferase disclosed by the invention can be used for synthesizing flavones such as t he wogonin, the isowogonin and the moslosooflavone, and can be used for catalyzing the synthesis and methylation reaction of flavonoid substances with a similar structure; and a foundation is laid for industrial large-scale fermentation production or enzymatic conversion method production of methylated flavonoid compounds.

Description

technical field [0001] The present invention relates to the field of biotechnology, specifically, the present invention relates to five flavone-8-methyltransferases, two flavone-7-methyltransferases and their functions in flavones such as wogonin, isowogonin and scutellaria The application in the synthesis of flavones and the like also relates to a method for using these enzymes to biosynthesize methylated flavones. Background technique [0002] Wogonin (5,7-dihydroxy-8-methoxyflavone), isowogonin (5,8-dihydroxy-7-methoxyflavone), scutellaria flavone (5-hydroxy-7, 8-dimethoxyflavone) (the structures of the three flavones are shown below) are the active ingredients of traditional Chinese herbal medicines Scutellaria baicalensis and Scutellaria barbata, which have strong inhibitory effects on key targets of cancer. The methylation at the 8-position and 7-position is the main structural feature of the above compounds. In the biosynthesis process of plant natural products, the...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12P17/06
CPCC12N9/1007C12P17/06
Inventor 江会锋刘晓楠程健卢丽娜
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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