Flavone synthase and application thereof

A flavonoid and enzyme activity technology, applied in the field of biological enzymes, can solve the problem that the conversion efficiency of FNSI cannot meet the requirements, and achieve the effect of optimal catalytic efficiency

Active Publication Date: 2019-12-27
TF BIOSYN BIOTECHNOLOGY CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After comparative analysis of literature, the catalytic efficiency of most FNS I is higher than that of FNS II (Effendi Leonard, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2005), but the conversion efficiency of existing types of FNS I still cannot meet the requirements of production and scientific research.

Method used

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  • Flavone synthase and application thereof
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  • Flavone synthase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1. Cloning of flavone synthase DcFNS and its expression in Escherichia coli

[0025] Two primers such as SEQ ID NO:5 and SEQ ID NO:6 in the sequence listing were synthesized, and the cDNA obtained by reverse transcription of RNA extracted from plants was used as a template to perform PCR using the above primers. The DNA polymerase was selected from the high-fidelity KOD DNA polymerase of Treasure Bioengineering Co., Ltd. The PCR amplification program was as follows: 94°C for 2min; 94°C for 15s, 58°C for 30s, 68°C for 2min, a total of 35 cycles; 68°C for 10min to 10°C. The PCR products were detected by agarose gel electrophoresis, and the results are shown in the attached figure 1 .

[0026] Under UV irradiation, the target DNA band is excised. Then Axygen Gel Extraction Kit (AEYGEN Company) was used to recover DNA from the agarose gel, which was the amplified DNA fragment of the flavone synthase gene. The recovered PCR product was cloned into the PMDT vector...

Embodiment 2

[0031] Embodiment 2. Flavone synthase DcFNS catalyzes naringenin reaction

[0032] Using the supernatant of BL21-pET28a-DcFNS and BL21-pET28a lysate obtained in Example 1 as the crude enzyme solution, configure the following reaction system (100 μL):

[0033]

[0034] React in a water bath at 30°C for 2h. After the reaction was completed, an equal volume of ethyl acetate was added for extraction, and the upper ethyl acetate phase was taken. After vacuum concentration, the reaction product was dissolved in 100 μL of methanol, and the results were detected by HPLC. The results are shown in the attached image 3 .

[0035] from image 3 From the results, it can be seen that the Escherichia coli crude enzyme solution BL21-pET28a-DcFNS containing flavone synthase DcFNS can catalyze naringenin to form a new product, and its retention time in HPLC is consistent with that of the standard apigenin, while the control group contains empty The Escherichia coli crude enzyme solution ...

Embodiment 3

[0036] Example 3. Cloning of flavone synthase AgFNS and its expression in Escherichia coli

[0037] Two primers such as SEQ ID NO: 9 and SEQ ID NO: 10 in the sequence listing were synthesized, and the cDNA obtained by reverse transcription of RNA extracted from plants was used as a template to perform PCR using the above primers. The DNA polymerase was selected from the high-fidelity KOD DNA polymerase of Treasure Bioengineering Co., Ltd. The PCR amplification program was as follows: 94°C for 2min; 94°C for 15s, 58°C for 30s, 68°C for 2min, a total of 35 cycles; 68°C for 10min to 10°C. The PCR products were detected by agarose gel electrophoresis, and the results are shown in the attached figure 1 .

[0038] Under UV irradiation, the target DNA band is excised. Then Axygen Gel Extraction Kit (AEYGEN Company) was used to recover DNA from the agarose gel, which was the DNA fragment of the amplified flavone synthase gene. The recovered PCR product was cloned into a PMDT vecto...

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Abstract

The invention provides flavone synthase and application thereof. The flavone synthase is selected from (a) polypeptide with an amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2, or (b) polypeptide formed by substitution, deletion, or addition of one or more amino acid residues of the amino acid sequence in SEQ ID NO:1 or SEQ ID NO:2, with the activity of the flavone synthase, and derived from (a), or (c) polypeptide with a sequence at least 85% identical to the amino acid sequence in SEQ ID NO:1 or SEQ ID NO:2 and the activity of the flavone synthase, and derived from (a). The flavone synthase can realize conversion and preparation of a plurality of flavone compounds, including apigenin, the better catalytic efficiency is achieved, the conversion efficiency better than that of existing FNS I is achieved, and the flavone synthase can be used for construction and optimization of flavone compound cell factories.

Description

technical field [0001] The invention relates to the technical field of biological enzymes, in particular to a flavone synthase and the application of the flavone synthase. Background technique [0002] Flavonoids are important plant natural compounds with high structural diversity. At present, more than 9,000 flavonoids with different structures have been isolated, which can be divided into flavones, flavanones, and isoflavones according to their structures. , flavanols, flavonols and anthocyanidines. Among them, flavonoids are the largest type of flavonoids. Flavonoids not only have a wide range of uses for plant physiology, ecology and agriculture, but also have significant therapeutic effects in the prevention and treatment of cancer and cardiovascular diseases. For example, apigenin (apigenin) is a flavonoid compound distributed in some warm-tropical vegetables and fruits, especially in celery. Apigenin has various biological activities such as anti-tumor, anti-inflam...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12P17/06
CPCC12N9/0071C12Y114/20C12P17/06
Inventor 周志华周金林叶德晓王平平严兴
Owner TF BIOSYN BIOTECHNOLOGY CO LTD
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