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Pichia pastoris expressing flavone synthase

A technology of Pichia pastoris and flavonoids, which is applied in the direction of oxidoreductase, microbial-based methods, and the use of vectors to introduce foreign genetic material, etc., can solve the problem that the conversion efficiency of FNSI cannot meet the requirements, and achieve the effect of stable expression and high efficiency

Active Publication Date: 2019-12-27
TF BIOSYN BIOTECHNOLOGY CO LTD
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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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  • Pichia pastoris expressing flavone synthase
  • Pichia pastoris expressing flavone synthase
  • Pichia pastoris expressing flavone synthase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1. Cloning of flavonoid synthase DcFNS and its expression in Escherichia coli

[0027] 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, and PCR was performed using the above primers. The DNA polymerase was the high-fidelity KOD DNA polymerase from Bao Bioengineering Co., Ltd. The PCR amplification program was: 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. PCR products were detected by agarose gel electrophoresis, and the results were as attached figure 1 .

[0028]Under UV irradiation, the target DNA band is excised. Then, using Axygen Gel Extraction Kit (AEYGEN Company) to recover DNA from the agarose gel, that is, the amplified DNA fragment of the flavonoid synthase gene. Using the PMD18-T cloning kit of Takara Bioengineering (Dalian) Co., Ltd. (Takara), the...

Embodiment 2

[0033] Example 2. Cloning of flavonoid synthase AgFNS and its expression in Escherichia coli

[0034] 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, and PCR was performed using the above primers. The DNA polymerase was the high-fidelity KOD DNA polymerase from Bao Bioengineering Co., Ltd. The PCR amplification program was: 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. PCR products were detected by agarose gel electrophoresis, and the results were as attached figure 1 .

[0035] Under UV irradiation, the target DNA band is excised. Then, using Axygen Gel Extraction Kit (AEYGEN Company) to recover DNA from the agarose gel, that is, the amplified DNA fragment of the flavonoid synthase gene. Using the PMD18-T cloning kit of Takara Bioengineering (Dalian) Co., Ltd. (Takara), t...

Embodiment 3

[0040] Example 3. Expression of flavonoid synthase DcFNS in Pichia pastoris

[0041] Synthesize two primers such as SEQ ID NO: 13 and SEQ ID NO: 14 in the sequence listing, in the synthesized primer, add two restriction sites of Sal I and Not I respectively at both ends, and use the plasmid obtained in Example 1 PCR was performed with PMDT-DcFNS as template. The PCR amplification procedure was the same as above. The PCR products were separated by agarose gel electrophoresis, and the recovered PCR products were ligated into the pPIC9K vector double digested by Sal I and Not I using Vazyme's One-step mutis clone kit. The obtained recombinant plasmid was named pPIC9K-DcFNS.

[0042] The recombinant plasmid pPIC9K-DcFNS was transformed into Escherichia coli Top10 to construct recombinant Escherichia coli Top10-pPIC9K-DcFNS.

[0043] The recombinant plasmid pPIC9K-DcFNS was extracted from Top10-pPIC9K-DcFNS, the recombinant plasmid pPIC9K-DcFNS and the empty plasmid pPIC9K were ...

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Abstract

The invention provides pichia pastoris expressing flavone synthase. An expression vector is contained, and the expression vector contains polynucleotide shown in the SEQ ID NO: 3 or the SEQ ID NO: 4.The pichia pastoris is disclosed to express FNS I (the flavone synthase), the advantages of stable expression and high efficiency are achieved, and the pichia pastoris can be applied to the 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 Pichia pastoris expressing flavonoid synthase. Background technique [0002] Flavonoids are important natural plant 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, isoflavones ( isoflavone ), 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, which is distributed in some vegetables and fruits in warm tropics, especially in celery. Apigenin has a variety of biological activities such as anti-tumor, anti-inflammatory and antioxidant. Its...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12N15/66C12N9/02C12R1/84
CPCC12N9/0004C12N15/815C12N15/66
Inventor 周金林周志华叶德晓王平平严兴
Owner TF BIOSYN BIOTECHNOLOGY CO LTD
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