Sesquiterpene cyclase, preparation and application thereof, and synthesis method of 2Z,4E-alpha-ionylideneehane

A technology of terpene cyclase and application method, applied in the fields of microbial chemistry and natural product chemistry, can solve the problems of high cost, low yield, low efficiency of natural ABA and the like

Active Publication Date: 2018-11-06
CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The chemical synthesis of the prior art is a mixture of two optical isomers (±)-ABA, in which the yield of (+)-ABA with biological activity is low, th

Method used

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  • Sesquiterpene cyclase, preparation and application thereof, and synthesis method of 2Z,4E-alpha-ionylideneehane
  • Sesquiterpene cyclase, preparation and application thereof, and synthesis method of 2Z,4E-alpha-ionylideneehane
  • Sesquiterpene cyclase, preparation and application thereof, and synthesis method of 2Z,4E-alpha-ionylideneehane

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Cloning of the gene encoding the sesquiterpene cyclase BcABA3.

[0048] 1. Culture of B.cinerea TBC-20 bacteria

[0049] Take a single colony slant of TBC-20 (the height of the slant is about 8cm, at 26°C, and the PDA medium is cultured for 7 days and then stored at 4°C), suck about 5ml of sterilized water with a dropper, and scratch the spores on the slant. Mix the mixture of spores several times, pipette 2ml into a 250ml Erlenmeyer flask containing 50ml of culture medium with a dropper, and place it on a shaker at 26°C and cultivate at 180rpm. After culturing for about 50h, the seed liquid was inoculated into a 250ml Erlenmeyer flask containing 50ml of culture medium with a 5ml pipette at a ratio of 5% of the inoculum, and cultured at 26°C and 180rpm. The medium consists of 6.0g / L glucose, 10g / L yeast extract, 3.0g / L soluble starch, 1.0g / L soybean flour, 2.0g / L sucrose, 0.5g / L NH 4 NO 3 and 1.0g / L KH 2 PO 4 composition.

[0050] 2. Extraction of genomic DNA from...

Embodiment 2

[0064] Construction of expression vector for sesquiterpene cyclase BcABA3.

[0065] The endonuclease cleavage sites NcoI and XhoI contained in the base sequence of the sesquiterpene cyclase BcABA3 obtained in Example 1 were determined.

[0066] The pET28a(+) plasmid (stored in the laboratory) was selected as the vector required for expressing the sesquiterpene cyclase BcABA3. Compare the enzyme cleavage site contained in the corresponding base sequence of the sesquiterpene cyclase BcABA3 with the enzyme cleavage site in the multi-cloning site region of the vector, and select the multi-cloning site region of the expression vector that contains the cleavage site. The NcoI and XhoI sites that are not in the base sequence of hemiterpene cyclase BcABA3 are used as restriction sites for constructing plasmids. See Table 2 for primers designed to contain restriction sites. PCR amplification was performed using KODplus-neo enzyme (purchased from Toyobo Co., Ltd.).

[0067] Table 2 S...

Embodiment 3

[0072] Linearization of the E. coli expression plasmid and transformation of E. coli to obtain a transformant containing the gene whose base sequence is shown in SEQ ID No. 1.

[0073] The recombinant plasmid pET-28(a)-SC3 obtained in Example 2 was transformed into competent cells of Escherichia coli E.coli BL21(DE3) strain (preserved in the laboratory) (the competent cells were prepared by the Inoue method). The specific operations are as follows:

[0074] 1) Pick a small amount of Transgene frozen E.coli DH5α cells stored at -80°C with a Tip, then streak the LB plate without antibiotics, and place the plate upside down in a bacterial incubator for 16 hours.

[0075] 2) Pick a newly activated E.coli DH5α single colony (2-3mm in diameter) from the LB plate and inoculate it in 3mL SOB liquid medium (add MgCl before use) 2 ), 37°C, 250rpm shaking culture for 7 hours, record the OD value.

[0076] 3) The bacterial suspension grown to the late logarithmic growth phase was inocul...

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Abstract

The invention discloses sesquiterpene cyclase, preparation and application thereof, and a synthesis method of 2Z,4E-alpha-ionylideneehane. At first, the amino acid sequence of provided sesquiterpene cyclase BcABA3 is represented by the sequence 2, and a derived polypeptide is also provided. The sesquiterpene cyclase can catalyze farnesyl pyrophosphate cyclization to synthesize 2Z,4E-alpha-ionylideneehane. At the same time, gene sequence that encodes the amino acid sequence of sesquiterpene cyclase is provided. A recombinant protein containing the amino acid sequence is also provided. The invention further provides a method of using the sesquiterpene cyclase, the recombinant protein, and farnesyl pyrophosphate taken as the substrate to synthesize 2Z,4E-alpha-ionylideneehane and abscisic acid. The invention further provides a recombinant expression carrier containing a sesquiterpene cyclase coding gene, a converter, and applications thereof. A synthesis method of 2Z,4E-alpha-ionylideneehane is also provided; according to the synthesis method, farnesyl pyrophosphate is taken as the substrate, and the synthesis is catalyzed by sesquiterpene cyclase or recombinant sesquiterpene cyclase.

Description

technical field [0001] The present invention relates to a sesquiterpene cyclase, its encoding gene, its application and its preparation, and relates to a method for synthesizing 2Z,4E-α-pyrene ethane using farnesyl pyrophosphate as a substrate. It belongs to the field of microbial chemistry and natural product chemistry. Background technique [0002] Abscisic acid (ABA) is a plant signaling molecule with a sesquiterpene structure, and is one of the five types of endogenous growth regulators in plants recognized internationally. It has two optical isomers. The natural ABA present in plants is basically dextrorotatory (+)-ABA ( figure 1 ). [0003] Natural ABA has great foundation and application value in cell engineering, agricultural production, ecological construction, landscaping and other fields, but because its content in plants is very low, artificial synthesis is an important source. The chemical synthesis in the prior art is a mixture of two optical isomers (±)-ABA...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12P5/02C12P7/42
CPCC12N9/001C12P5/026C12P7/42
Inventor 舒丹谭红周金燕丁忠涛钟娟罗笛杨杰
Owner CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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