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NADPH-cytochrome P450 reducing ferment and application thereof

A technology of nicotinamide adenine and cytochrome, which is applied in the fields of biotechnology and plant biology, can solve the problems that the catalytic activity needs to be improved and the kinship is far away.

Active Publication Date: 2015-09-09
SYNBIOTECH (SUZHOU) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these ginseng-derived CYPs also have catalytic activity in combination with CPR derived from Arabidopsis thaliana, their catalytic activity needs to be improved due to the distant relationship between Arabidopsis and ginseng

Method used

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  • NADPH-cytochrome P450 reducing ferment and application thereof
  • NADPH-cytochrome P450 reducing ferment and application thereof
  • NADPH-cytochrome P450 reducing ferment and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Embodiment 1, the cloning of nicotinamide adenine dinucleotide (NADPH)-cytochrome P450 reductase

[0062] The four primers synthesized respectively have the nucleotide sequences of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 in the sequence listing.

[0063] Using the cDNA obtained by reverse transcription of RNA extracted from ginseng as a template, PCR was performed using the above two pairs of primers SEQ ID NO: 3 / 4 and SEQ ID NO: 5 / 6, respectively. 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, then drop to 10°C. The PCR products were detected by agarose gel electrophoresis, and the results were as follows: figure 1 .

[0064]Under UV irradiation, the target DNA band is excised. Then, Axygen Gel Extraction Kit (AXYGEN Company) was used to recover DN...

Embodiment 2

[0067] Example 2, Construction of recombinant vectors expressing PgDDS, CYP716A47, PgCPR1 and PgCPR2 genes

[0068] (1) Synthesizing two primers respectively having the nucleotide sequences of SEQ ID NO:7 and SEQ ID NO:8 in the sequence listing.

[0069] Two restriction sites, BamH I and Xho I, were respectively set at both ends of the synthetic primers SEQ ID NO:7 and SEQ ID NO:8 (amplification of CYP716A47), and PCR was performed using ginseng cDNA as a template. The PCR amplification procedure is the same as in Example 1. The PCR product was separated and recovered by agarose gel electrophoresis, and after being digested by BamH I and Xho I, the T4 DNA ligase of NEB Company was used to connect into the pESC-HIS vector (Agilent Technologies). The obtained recombinant plasmid was named pESC-HIS-CYP.

[0070] (2) Synthesizing six primers respectively having the nucleotide sequences of SEQ ID NO: 11-16 in the sequence listing. Using the genome of Saccharomyces cerevisiae BY...

Embodiment 3

[0080] Example 3. In vitro NADPH-cytochrome P450 reductase and cytochrome P450 (CYP716A47) synergistically catalyze the conversion of dammarenediol to protopanaxadiol

[0081] (1) The recombinant plasmid pHCR1 was introduced into Saccharomyces cerevisiae BY4742 (purchased from Euroscarf) using the Frozen-EZ Yeast Transformation II Transformation Kit of ZYMO Research Company to construct recombinant Saccharomyces cerevisiae BY-CYP-CPR1. Prepare liquid induction medium: 0.67% (w / v) yeast nitrogen source (without amino acids), 2% (w / v) galactose, 0.01% (w / v) leucine, 0.01% (w / v) lysine amino acid, 0.01% (w / v) uracil. Inoculate BY-CYP-CPR1 in 50ml induction medium to induce culture for four days. After collecting the cells by centrifugation, the cells were lysed at low temperature and centrifuged in an ultracentrifuge to prepare microsomes. The recombinant plasmid pESC-HIS-CYP was introduced into BY4742 in the same way to construct recombinant Saccharomyces cerevisiae BY-CYP (bl...

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Abstract

Provided is a nicotinamide adenine dinucleotide (NADPH)-cytochrome P450 reductase and a use thereof. Also provided is a polynucleotide encoding the NADPH-cytochrome P450 reductase, an expression vector and a host cell which express the NADPH-cytochrome P450 reductase, and a method for producing the protopanoxadiol.

Description

technical field [0001] The present invention relates to the field of biotechnology and plant biology; more specifically, the present invention relates to nicotinamide adenine dinucleotide (NADPH)-cytochrome P450 reductase and application thereof. Background technique [0002] Nicotinamide adenine dinucleotide (NADPH)-cytochrome P450 reductase (EC1.6.2.4Cytochrome P450 Reductase, CPR) is an important part of the cytochrome P450 (CYP) catalytic system. The redox reaction catalyzed by cytochrome P450 requires the supply of electrons (reducing power). NADPH-cytochrome P450 reductase can provide reducing power for the reaction catalyzed by CYP by obtaining electrons from electron donors and transferring them to CYP. In the absence of CPR, CYP has no catalytic activity on the substrate. NADPH-cytochrome P450 reductase belongs to the riboflavin protein family, is a membrane protein and has a conserved domain that binds to the coenzymes FMN, FAD and NADPH. [0003] Since the domai...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/63C12P33/00
CPCC12N9/0073C12P33/06C12Y114/13
Inventor 周志华王平平严兴范云
Owner SYNBIOTECH (SUZHOU) CO LTD
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