Flavones-6-hydroxylase and application thereof to scutellarin synthesis

A kind of hydroxylase and flavonoid technology, which is applied to flavonoid-6-hydroxylase and its application field in the synthesis of scutellarin

Active Publication Date: 2017-01-11
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] At present, there is no relevant report on flavone 6-hydroxylase derived from scutellaria, so there is an urgent need in this field to develop a method for naturally synthesizing scutellarein and scutellarin

Method used

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  • Flavones-6-hydroxylase and application thereof to scutellarin synthesis
  • Flavones-6-hydroxylase and application thereof to scutellarin synthesis
  • Flavones-6-hydroxylase and application thereof to scutellarin synthesis

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preparation example Construction

[0128] The preparation method of scutellarin

[0129] The present invention also provides a preparation method of scutellarin, comprising the steps of:

[0130] (i) in the presence of cytochrome reductase (CPR), utilize flavone-6-hydroxylase to catalyze apigenin, thereby obtaining scutellarein;

[0131] with

[0132] (ii) performing a glycosylation reaction on the scutellarein obtained in step (i), thereby obtaining scutellarin;

[0133]

[0134] The main advantages of the present invention include:

[0135] (1) The present invention finds for the first time that flavone-6-hydroxylase can catalyze the hydroxylation of C6 of apigenin, thereby obtaining scutellarein.

[0136] (2) The present invention provides for the first time flavone-6-hydroxylase derived from scutellaria breviscapus, and finds that the flavone-6-hydroxylase derived from scutellaria breviscapus can catalyze the C6 of apigenin to carry out the catalytic reaction of hydroxylation, thereby obtaining wild...

Embodiment 1

[0142] Example 1 Obtaining of EbF6H Encoding Nucleotides

[0143] According to the nucleotide sequence information (SEQ ID NO.: 2) provided by the present invention, it is obtained by amplifying or isolating the RNA reverse transcriptome of Breviscapus breviscapus. Specifically, RNA can be extracted from plants using conventional methods or using the Easy spin plus plant RNA rapid extraction kit (purchased from Beijing Aidelai Biotechnology Co., Ltd.), and then the obtained RNA is used as a template to synthesize cDNA using First Strand The kit (Thermo, USA) reverse-transcribed the cDNA obtained according to the operation steps on the kit, and used it as a template, using primers EbF6H-F1 (SEQ ID NO: 9) and EbF6H-R1 (SEQ ID NO: 10) using conventional The PCR method specifically amplifies the coding nucleotide of EbF6H. PCR amplified fragments were subjected to agarose gel electrophoresis, the results were as follows figure 2 shown.

Embodiment 2

[0144] The acquisition of the coding nucleotide of embodiment 2 CcsF6H

[0145] Using the amino acid sequence of EbF6H of Erigeron breviscapus to compare with other Asteraceae species, it is found that there is an amino acid sequence with about 69% similarity with EbF6H in Artichoke, another Asteraceae species whose genome has been sequenced, and its Genbank number is KVH90146.1. After identification, it was deduced that this gene also has a similar function of flavonoid 6-hydroxylation, and it was named CcsF6H.

[0146] According to the amino acid sequence of SEQ ID NO: 3, the nucleotide sequence codon is optimized for the target host cell (Saccharomyces cerevisiae) and artificially synthesized to obtain the sequence shown in SEQ ID NO: 6. Using CcsF6H-F1 (SEQ ID NO: 11) and CcsF6H-R1 (SEQ ID NO: 12) as primers, a conventional PCR method was used to specifically amplify the coding nucleotide of CcsF6H. PCR amplified fragments were subjected to agarose gel electrophoresis, t...

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Abstract

The invention provides flavones-6-hydroxylase and application thereof to scutellarin synthesis and particularly provides flavones-6-hydroxylase which is capable of catalyzing apigenin 6-coordinate hydroxylation reaction to obtain scutellarein. In addition, the invention provides a method for preparing the scutellarein into scutellarin by adoption of glycosylated transferases and glycosyl donors.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular, the invention relates to flavone-6-hydroxylase and its application in the synthesis of scutellarin. Background technique [0002] Erigeron breviscapus (Vant.) Hand-Mazz is the dry whole herb of Compositae plant Erigeron breviscapus (Vant.) Hand-Mazz. It is cold in nature, slightly bitter, sweet and warm. Active, anti-inflammatory and analgesic effects. Erigeron breviscapus medicines have remarkable clinical application effects, and are listed as nationally developed Chinese herbal medicine varieties and clinically necessary emergency medicines for the treatment of cardiovascular and cerebrovascular diseases by traditional Chinese medicine. In addition to being mainly used for diseases of the cardiovascular and cerebrovascular systems in clinical practice, Erigeron Injection also has good curative effects in the treatment of diabetes, nephropathy, cervical vertigo, and senile diseases....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12P17/06C12P19/60
Inventor 江会锋刘晓楠丁文涛程健段立津阮江星卢丽娜
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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