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Flavone synthase I/flavanone-3-hydroxylase and application thereof in field of flavonoid compound synthesis

A flavanone, hydroxylase technology, applied in the directions of application, microorganism-based method, angiosperms/flowering plants, etc., can solve problems such as flavonoid synthase I and flavanone-3-hydroxylase have not been reported yet

Active Publication Date: 2022-08-05
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Ferns are rich in flavonoids with important pharmacological activities, but their flavone synthase I and flavanone-3-hydroxylase have not been reported
However, there is no research report on the key enzymes involved in the biosynthesis of flavonoids in pine fern

Method used

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  • Flavone synthase I/flavanone-3-hydroxylase and application thereof in field of flavonoid compound synthesis
  • Flavone synthase I/flavanone-3-hydroxylase and application thereof in field of flavonoid compound synthesis
  • Flavone synthase I/flavanone-3-hydroxylase and application thereof in field of flavonoid compound synthesis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1 Cloning of the expression gene PnFNS I / F3H

[0061] 1.1 CTAB-PVP method to extract total RNA of P.

[0062] The preparation method of CTAB-PVP extraction buffer is as follows:

[0063] 100 mM Tris·HCl (pH 8.0), 2% CTAB (w / v), 2% PVP (w / v), 25 mM EDTA, 2 M NaCl, mercaptoethanol was added to 0.2% after autoclaving; solution preparation was treated with DEPC ddH 2 O, spare after autoclaving.

[0064] Extraction Method:

[0065] (1) The water bath was adjusted to 65°C, and the prepared CTAB extract was placed in it to preheat. Take an appropriate amount of material into a mortar, add liquid nitrogen and grind into powder.

[0066] (2) Put the ground material into a liquid nitrogen quick-frozen imported 2 mL centrifuge tube, add 600 μL of CTAB extract, and invert up and down to mix evenly.

[0067] (3) 65 ℃ water bath, mix once every 10 minutes, and heat for 30 minutes.

[0068] (4) Take out the centrifuge tube and wait for the temperature to drop to room te...

Embodiment 2

[0120] Example 2 Gene protein expression and enzyme activity function analysis

[0121] 2.1 Construction of protein expression vector

[0122] 2.1.1 Amplify the target gene

[0123] The correctly sequenced monoclonal bacteria were cultured overnight at 37°C at 120rpm, and the plasmid was extracted using the Plasmid Mini Kit:

[0124] (1) The existing strains MemUGT1-pTOPO-DH5α were divided into LB plates (containing 100 μg / mL Amp), 37 ° C, after 12 h, single clones were grown, and single clones were picked in 4 mL of Amp-resistant medium, 37 ° C , 110rpm cultured for 10h.

[0125] (2) Centrifuge the bacterial solution at 12,000 rpm for 1 min at room temperature, discard the supernatant, collect the bacterial cells, and discard the supernatant as much as possible.

[0126] (3) Add 150 μL of solution P1 to the centrifuge tube with the bacterial cell precipitation left, and vortex to shake until the bacterial cell is completely suspended.

[0127] (4) Add 150 μL of solution P...

Embodiment 3

[0186] Example 3 Biosynthesis of apigenin and dihydrokaempferol using Escherichia coli PnFNS I / F3H-pET32a-BL21.

[0187] (1) Activate the strain in a constant temperature incubator at 37 °C, pick a single clone and inoculate it into 4 mL of LB liquid medium (containing 100 μg / mL of Amp), and continue to cultivate in a 37 °C incubator for 7 hours;

[0188] (2) The target strain and the control strain were inoculated into 50 mL of resistant LB medium according to the ratio of 1:100, and cultured at 37°C in a shaker at 200 rpm to OD600=0.6-0.8, and IPTG was added to make the final concentration 0.5 mM , 20 ℃ constant temperature incubation 6-7h;

[0189] (3) Add the DMSO-dissolved substrate (naringenin) to the bacterial solution, the substrate concentration is 150 μM, and put it into 20°C to continue culturing for a period of time;

[0190] (4) Take out 500μL of bacterial liquid every 12h, add an equal volume of ethyl acetate to extract 2-3 times, combine the organic phases, blo...

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Abstract

The invention relates to a flavone synthase I / flavanone-3-hydroxylase and application of the flavone synthase I / flavanone-3-hydroxylase in the field of flavonoid compound synthesis. The invention discloses flavone synthase I / flavanone-3-hydroxylase of psidium caudatum. The amino acid sequence of the flavone synthase I / flavanone-3-hydroxylase of the psidium caudatum is as shown in SEQ ID No. 1. According to the invention, a bifunctional enzyme capable of introducing a double bond between a flavanone C2 site and a flavanone C3 site or introducing a hydroxyl group at the flavanone C3 site is cloned and identified from ferns for the first time. The gene has relatively high catalytic activity on common flavanone compounds, and a candidate gene is provided for producing the flavanone compounds. The PnFNS I / F3H can be used for synthesizing flavones and flavanonol in escherichia coli, and the contents of plant flavones and flavanonol compounds in transgenic arabidopsis thaliana are increased at the same time. The gene can be used for producing flavone and flavonol compounds in escherichia coli and plant chassis, and has high application value.

Description

technical field [0001] The invention belongs to the technical field of flavonoid compound catalytic enzymes, and in particular relates to a flavonoid synthase I / flavanone-3-hydroxylase derived from Pteridophyta, a nucleic acid substance encoding the enzyme, an expression vector comprising the nucleic acid substance, A host cell, an engineered bacterium expressing the flavonoid synthase I / flavanone-3-hydroxylase and its application in the field of flavonoid synthesis. Background technique [0002] The disclosure of information in this Background section is only for enhancement of understanding of the general background of the invention and should not necessarily be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art. [0003] As a class of important secondary metabolites, flavonoids are widely found in terrestrial plants. Studies have shown that flavonoids have various biologic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/70C12N1/21C12N15/84A01H5/10A01H5/00A01H6/20C12P17/06C12R1/19
CPCC12N9/0071C12N15/70C12N15/8243C12N15/8205C12P17/06C12Y114/20C12Y114/11009Y02A50/30
Inventor 程爱霞傅杰谭慧汪飘逸娄红祥
Owner SHANDONG UNIV
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