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Alpha-santalene synthetase, gene and application

A technology of santalene and synthetase, which is applied in the fields of application, genetic engineering, plant gene improvement, etc., and can solve the problem of low product specificity

Active Publication Date: 2021-06-11
HANGZHOU NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are few studies on santalene synthase. For example, santalene synthase was cloned from Santalum album, S. austrocaledonicum and S. spicatum respectively, but the product specificity is not high, except for the generation of α / In addition to β-santalene, there are also its isomers epi-β-santalene, bergamotene and other products

Method used

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  • Alpha-santalene synthetase, gene and application
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  • Alpha-santalene synthetase, gene and application

Examples

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Effect test

Embodiment 1

[0019] Example 1: Preparation of α-santalene synthase, gene, carrier, and engineering bacteria derived from curcuma curcuma

[0020] 1. Homology analysis of candidate CwSS genes

[0021] The roots, young leaves, buds and stems of Curcuma curcuma were collected from both sides of the Feiyun River in Ruian, Wenzhou, and the transcriptome sequencing was performed by Shanghai Meiji Biotechnology Company. The obtained sequences were analyzed by bioinformatics and found a candidate sesquiterpene synthase gene ( That is, the α-santalene synthase (CwSS) of the present application, its deduced amino acid sequence was analyzed by Blastp at NCBI and found to have the greatest identity with the bisabolene synthase (Accession Number: D2YZP9) derived from Zingiber officinale. sequence identity of 86%; further sequence comparison with sesquiterpene synthases from other sources shows that CwSS has conserved domains, such as DDxxD, RXR, NSE / DTE, RRX 8 W( figure 1 ).

[0022] Phylogenetic tr...

Embodiment 2

[0028] Embodiment 2: Analysis of catalytic properties of recombinant enzyme CwSS

[0029] Using the CwSS recombinant enzyme solution prepared by the method in Example 1 (concentration: 50 mg / L, volume: 10 μL) as a catalyst, add pH 7.0 Tris-HCl buffer solution, 2 μg FPP (farnesyl pyrophosphate), 500 mM MgCl 2 20 μL of the solution, 2 μL of 500 mM DTT, and 100 μL of dilute glycerol (1,2,3-propanetriol) constituted a reaction system of 1 ml, which were stirred and fully reacted at 30°C for 120 minutes. After the reaction is over, the extraction head is taken out and injected into a gas chromatograph for qualitative analysis of the product. Chromatographic conditions: GC-2010 Shimadzu gas chromatograph is selected; the chromatographic column is HP-5; carrier gas: N 2 , the purge flow rate is 3mL / min, without splitting; the initial temperature of the column oven is 40°C, hold for 2 minutes, and then increase the temperature to 220°C at a speed of 7°C / min, and hold for 5 minutes; t...

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Abstract

The invention discloses an alpha-santalene synthase, a gene and application. The alpha-santalene synthetase is derived from Curcuma wenyujin Y.H.Chen et C.Ling, and the amino acid sequence of the alpha-santalene synthetase is as shown in SEQ ID NO. 2. Functional verification is carried out on alpha-santalene synthetase from curcuma wenyujin, and an alpha-santalene synthesis method is invented and applied to escherichia coli. According to the present invention, it is found that the curcuma wenyujin-sourced alpha-santalene synthase can catalyze FPP in a prokaryotic expression system to generate a large amount of alpha-santalene and a small amount of beta-santalene and alpha-farnesene, and the content of the alpha-santalene calculated according to the peak area achieves more than 90%, such that the catalyst can be rapidly and massively prepared in Escherichia coli and be applied to synthesis of alpha-santalene, and the catalyst can be further applied to synthesis of alpha-santalol and has important significance.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an α-santalene synthase, gene and application. Background technique [0002] α-santalene (α-santalene), whose structural formula is shown in formula I below, is a sesquiterpene volatile component extracted from sandalwood, which can generate α-santalol under the catalysis of cytochrome oxidase P450, α- Santalene and α-santalol are the main components of sandalwood essential oil. Sandalwood essential oil has low toxicity and no mutagenicity, and is currently recognized as a safe food additive; sandalwood essential oil is not only used in spices and cosmetics, but also has sedative, anti-inflammatory, analgesic, antibacterial, antiviral, antioxidative, and antitumor effects , has a certain curative effect on skin diseases, bronchitis, mucositis, depression and insomnia. Although sandalwood essential oil plays a very important role and its market value is also expanding, sandalwood es...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21C12P15/00C12R1/19
CPCC12N9/88C12N15/70C12P15/00C12Y402/03082
Inventor 谢恬殷晓浦谌容卫秋慧刘雨恒魏昕
Owner HANGZHOU NORMAL UNIVERSITY
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