Novel triterpene synthase and application thereof

An amino acid and cell technology, applied in the fields of biotechnology and plant biology, can solve the problems of unseparated, unable to synthesize compounds in large quantities, hindering analysis and application, etc.

Pending Publication Date: 2022-04-05
CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, more than 100 kinds of triterpene skeletons have been identified in plants, including 85 triterpene synthases with different functions, and some biologically active triterpene compounds were isolated and iden...

Method used

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  • Novel triterpene synthase and application thereof
  • Novel triterpene synthase and application thereof
  • Novel triterpene synthase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Embodiment 1, the cloning of sagozaol synthase (ZmOSC1)

[0086] Two primers were synthesized to respectively have the nucleotide sequences of SEQ ID NO:3 and SEQ ID NO:4 in the sequence listing.

[0087] Using the cDNA obtained by reverse transcription of RNA extracted from corn as a template, PCR was performed using the above two pairs of primers SEQ ID NO:3 and SEQ ID NO:4. The DNA polymerase was selected from Takara Bioengineering (Dalian) Co., Ltd. (Takara) high-fidelity PrimeStar DNA polymerase. The PCR amplification program is: 98°C for 2min; 98°C for 10s, 58°C for 15s, 68°C for 3min, a total of 35 cycles; 68°C for 7min, then drop to 10°C. The PCR products were detected by agarose gel electrophoresis, and the results were as follows: figure 1 .

[0088] Under UV irradiation, the target DNA band is excised. Then Axygen Gel Extraction Kit (AXYGEN Company) was used to recover DNA from the agarose gel, which was the amplified DNA fragment of the triterpene syntha...

Embodiment 2

[0090] Example 2, Construction of Recombinant Saccharomyces cerevisiae WP7-pESC-Leu-ZmOSC1

[0091] 1. Establishment of engineered cells

[0092] Enhancing the ERG10, ERG13, ERG12, ERG8, ERG19, IDI and tHMG1 genes of yeast CENPK-II (purchased from Euroscarf), and overexpressing these genes in yeast CENPK-II to obtain high yields of squalene and 2,3 epoxy squalene Yeast-engineered cell WP7 of squalene. The sequence information of each gene is shown in Table 1.

[0093] 2. Establishment of recombinant Saccharomyces cerevisiae WP7-pESC-Leu-ZmOSC1

[0094] Two primers respectively having the nucleotide sequences of SEQ ID NO:5 and SEQ ID NO:6 in the sequence listing were synthesized.

[0095] Homology arm sequences were set at both ends of the synthesized primers SEQ ID NO:5 and SEQ ID NO:6 (amplification of ZmOSC1), respectively, and PCR was performed using cDNA reverse-transcribed from RNA extracted from corn as a template. The PCR amplification procedure is the same as in E...

Embodiment 3

[0097] Embodiment 3, utilizing recombinant Saccharomyces cerevisiae to induce the production of sago cuckoo alcohol

[0098] 1. Induction culture method

[0099] Pick the yeast streaked on the solid SCO medium plate: WP7-pESC-Leu-ZmOSC1 and WP7-pESC-Leu were shaken and cultured in test tubes containing 4mL liquid seed medium overnight (30°C, 250rpm, 16h); 1% of the inoculum was transferred to a 250ml Erlenmeyer flask containing a liquid synthetic medium for shake-flask fermentation, and cultured at 30° C. with shaking at 250 rpm for 4 days to obtain a fermentation product.

[0100] 2. Western blot analysis

[0101] Centrifuge at 13000g for 30 seconds to collect the cells, add pH8.0 Tris-HCl buffer to resuspend the cells, transfer to a Fastprep tube at 6M / s, shake for 35s / time for 4 times, centrifuge at 12000g at 4°C to collect the supernatant of the cell lysate In a new EP tube, centrifuge at 15000 for 20 minutes to collect the supernatant. Samples were taken for Western bl...

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Abstract

The invention provides a novel triterpene synthase as well as preparation and application thereof. The triterpenoid synthase provided by the invention can effectively catalyze a triterpenoid compound precursor to form simedolol. The triterpene synthase disclosed by the invention can be expressed in engineering cells such as yeast and the like, can be used for producing the rhododendron simsii alcohol synthase, and has a very good application prospect.

Description

technical field [0001] The invention relates to the fields of biotechnology and plant biology; more specifically, the invention relates to a novel triterpene synthase and its application. Background technique [0002] Triterpenoids are a large class of natural products that widely exist in plants. They have complex structures and diverse functions, and play important functions in many fields. Triterpene synthase, as a key enzyme in the synthesis pathway of triterpenoids, forms a core skeleton with diverse structures, which is the basis for the structural diversity of triterpenoids. [0003] Triterpene synthase catalyzes the formation of different carbon skeletons from 2,3 epoxy squalene, which is further modified by modifying enzymes such as cytochrome P450, glycosyltransferase and acyltransferase to produce triterpene compounds with diverse structures, which are widely used in Food, medicine and industrial biotechnology and other fields. Therefore, the exploration of trit...

Claims

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Application Information

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IPC IPC(8): C12N9/90C12N15/61C12N15/81C12N1/19C12P15/00C12R1/865
Inventor 周志华樊震鋆王燕严兴王平平
Owner CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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