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Indole synthase and its application

A technology for terpenes and sesquiterpenes, which is applied to terpenoid synthase and its application field, can solve the problems of increasing the difficulty of sesquiterpene skeleton and limiting the diversity of skeleton.

Active Publication Date: 2019-08-06
SHENZHEN ACTION TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since sesquiterpene compounds only contain 3 isoprene units, the length of the carbon chain limits the diversity of its skeleton, which also greatly increases the difficulty of mining new sesquiterpene skeletons

Method used

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  • Indole synthase and its application
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  • Indole synthase and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0058] In this example, the cDNA sequence of terpene synthase J1-018-A (abbreviated as J1-018-A) was amplified by PCR method, and the cDNA sequence was used to construct a plasmid and a high-yield sesquiterpene yeast platform, wherein, The strains and plasmids used in this example are shown in Table 1, and the primers used to construct related plasmids are shown in Table 2.

[0059] The main characteristics of the strains and plasmids used in Table 1

[0060]

[0061] Table 2 Primers used for plasmid construction

[0062]

[0063]

[0064]

[0065] 1. J1-018-A plasmid construction and protein purification

[0066] Using the reverse-transcribed F. graminearum J1-012 cDNA as a template, use primers P1 / P2 to amplify to obtain the cDNA sequence of J1-018-A, and then enzyme-digest and connect it to plasmid pET28a(+) to obtain plasmid pGB152, as follows:

[0067] (1) Obtaining cDNA: QIAGEN RNeasy Plant Mini Kit was used for fungal RNA extraction, and the operation was ...

Embodiment 2

[0117] In this example, the J1-018-A fermentation product obtained in Example 1 is purified and identified, as follows:

[0118] 1. Product purification of S.cerevisiae T16 mutant strain

[0119] Purified mobile phase A is ultrapure water, mobile phase B is acetonitrile, and the ultraviolet absorption wavelength is 210nm. The J1-018-A fermentation product is purified by semi-preparative HPLC according to the conditions described in Table 1, and the target product is determined by GC-MS According to the peak time on HPLC, the F1-F7 components containing sesquiterpene products were collected, among which, F1-F4 were the main sesquiterpene components. For details, see image 3 .

[0120] Purification conditions of compounds in table 1S.cerevisiae T16 mutant strain

[0121]

[0122] The analysis results show that the purity of compound 1 in component F4, compound 6 in component F6 and compound 8 in component F7 can be directly used for NMR detection without further purificati...

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Abstract

The invention discloses a terpene synthase and its application. Specifically, the invention provides use of the terpene synthase and a plasmid and a vector expressing the terpene synthase for catalyzing the synthesis of terpenes or preparing a catalyst for a reaction for catalyzing the synthesis of hydrazines, and also provides a method for the preparation of terpenes. The terpene synthase can specifically and efficiently synthesize a plurality of sesquiterpene compounds by using a terpenoid precursor material as a substrate.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to terpene synthase and its application. Background technique [0002] Sesquiterpene compounds are synthesized from two molecules of isopentenyl pyrophosphate (IPP) and one molecule of dimethylpropylene pyrophosphate (DMAPP) under the catalysis of farnesyl pyrophosphate synthase (farnesyl pyrophosphate synthase). Phosphate (farnesyl pyrophosphate, FPP), which is then formed under the catalysis of sesquiterpene synthase. So far, at least 122 different sesquiterpenoid skeletons have been discovered (Klapschinski et al., 2016). Some of the skeleton compounds, such as 5-7 double-membered rings and 3-5-6 three-membered ring sesquiterpene compounds, have various biological activities such as antibacterial and antitumor. However, since sesquiterpenoids only contain 3 isoprene units, the length of the carbon chain limits the diversity of their skeletons, which greatly increases the difficult...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12P5/00C12P5/02C12P7/02C12P7/04
CPCC12N9/1085C12P5/007C12P5/002C12P5/026C12P7/02C12P7/04C12P5/02C12N9/10C12P5/00C12N15/11C12N9/00Y02P20/584
Inventor 叶紫玲
Owner SHENZHEN ACTION TECH CO LTD
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