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Protopanoxadiol biosynthesizing method and bacterial strain for producing protopanoxadiol

A protopanaxadiol and synthase technology, applied in the field of genetic engineering, can solve the problems of complex extraction technology, long growth cycle, and low purity

Inactive Publication Date: 2013-06-26
QINGDAO VLAND BIOTECH GRP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In short, the acquisition of protopanaxadiol has difficulties such as long growth cycle, low raw material content, complex extraction technology, and low purity.

Method used

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  • Protopanoxadiol biosynthesizing method and bacterial strain for producing protopanoxadiol
  • Protopanoxadiol biosynthesizing method and bacterial strain for producing protopanoxadiol
  • Protopanoxadiol biosynthesizing method and bacterial strain for producing protopanoxadiol

Examples

Experimental program
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Effect test

Embodiment 1

[0041] Embodiment 1 Construction of dammarenediol synthase expression vector

[0042] The dammarenediol synthase gene GenBank: AB265170.1 in the ginseng genome was codon optimized according to the codon preference of Pichia pastoris GS115, and the optimized dammarenediol synthase gene sequence is SEQ ID NO: 1, SEQ ID NO:1 was synthesized by Shanghai Qinglan Biotechnology Co., Ltd.

[0043] Design restriction restriction sites AsuII and XbaI at both ends of the gene fragment. Using primer F respectively:

[0044] 5'-TACTTCGAAATGGCCTGGAAGCAAAAAAGGTGCTC-3' and R:

[0045] 5'-GCCTCTAGATTAAATTTTCAACTGCTGATGTTAG-3' was amplified by PCR. PCR amplification conditions: 94°C, 5min; 94°C, 30S; 58°C, 30S, 30 cycles; 72°C, 2.5min; 72°C, 10min.

[0046]The PCR amplified product and Pichia pastoris expression plasmid pPicZa were digested with AsuII and XbaI, and digested overnight at 37°C, and the digested product was recovered and purified on agarose gel. Design a ligation system with a...

Embodiment 2

[0047] Example 2 Construction of recombinant expression engineering bacteria for dammarenediol synthase

[0048] Positive transformants were cultured in LB medium at 37°C and 200rpm for 8h, the cells were collected, and the plasmid DS-pPICZa was extracted. SacI single-digestion treatment, overnight at 37°C, PCR purification and recovery, collected in 15ul of water, and stored at 4°C until use.

[0049] Prepare Pichia pastoris GS115 competent cells, add SacI-treated DS-pPICZa plasmid, mix well, place in ice bath for 5 minutes, transfer to electroporation cup, 1.98kv, 5.8ms, electric shock transformation. Add 1ml of sorbitol that was pre-cooled in advance, and incubate at 30°C for 30min. Collect the bacteria by centrifugation, add 1ml of YPD medium, rejuvenate at 200rpm at 30°C for 1 hour, collect the bacteria, smear on 100ug / ml bleomycin resistance screening YPD plate, culture at 30°C for 48h-72h. 800ug / ml bleomycin resistance was used to screen high-copy transformants, colon...

Embodiment 3P450

[0053] Example 3 Construction of P450 Cytochrome Synthetase Expression Vector

[0054] The P450 cytochrome synthase (CYP) gene GenBank: JN604537.1 in the ginseng genome was codon optimized according to the codon preference of Pichia pastoris. The optimized gene sequence is SEQ ID NO: 2, which was provided by Shanghai Qing Synthesized by Lan Biotechnology Co., Ltd.

[0055] The upstream and downstream restriction sites of the CYP gene fragment were designed to be BamHI and NotI, respectively. Design PCR primer F:

[0056] 5'-CAT GGATCC ATGGTTTTGTTCTTTTCCCTTTCAC-3' and R:5'-

[0057] GAAT GCGGCCGC TTAGTTATGAGGGTGAAGGTGAATAG-3'PCR amplification conditions are shown in the table below:

[0058]

[0059] The PCR product and its Pichia pastoris expression plasmid Ppic9k were digested with BamHI and NotI, and digested at 37°C for 4 hours. After the fragments were recovered by cutting the agarose gel, they were ligated overnight at 16°C. Prepare Escherichia coli DH5a compe...

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Abstract

The invention relates to a protopanoxadiol biosynthesizing method and provides a production bacterial strain of the protopanoxadiol. According to the protopanoxadiol biosynthesizing method, a pichia pastoris engineering strain constructed through a genetic engineering technological means can be used for efficiently co-expressing dammarendiol synthetase and P450 cytochrome synthetase, and further the protopanoxadiol is catalyzed and synthesized. The protopanoxadiol biosynthesizing method lays a foundation for large-scale industrial production of the protopanoxadiol.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a biosynthesis method of protopanaxadiol and a production strain thereof. technical background [0002] Protopanaxadiol (PPD) is a diol-type ginsenoside, mainly derived from Araliaceae plants such as Panax ginseng and American ginseng. Ginsenosides are tetracyclic triterpenoids with various pharmacological activities. According to their chemical structures, they can be divided into three types: protopanaxadiol type, protopanaxatriol type and oleanolic acid type. Protopanaxadiol is the aglycon of ginsenosides in the diol group, and is also the final product of ginsenosides in the diol group after microbial fermentation. There are 2 epimers, 20 (S) and 20 (R), and they all belong to Among the dammarane-type tetracyclic triterpenoids, 20(S)-protopanaxadiol is the most active. Pharmacological experiments have confirmed that 20(S)-protopanaxadiol can inhibit ...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12N15/53C12N15/60C12P33/00C12R1/84
CPCC12P33/00C12N9/0071C12N9/88
Inventor 王华明黄涛原静黄亦钧
Owner QINGDAO VLAND BIOTECH GRP
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