Search

CN-121991980-A - Wild jujube CYP450 enzyme gene ZjCYP13181 and application thereof in wild jujube saponin biosynthesis

CN121991980ACN 121991980 ACN121991980 ACN 121991980ACN-121991980-A

Abstract

The invention relates to a wild jujube CYP450 enzyme gene ZjCYP and 13181 and application thereof in wild jujube saponin biosynthesis. The nucleotide sequence of the gene is shown as SEQ ID NO.1, the total length of the sequence is 1554bp, the amino acid sequence of the encoded protein is shown as SEQ ID NO.2, and 517 amino acid residues are encoded. The wild jujube CYP450 enzyme gene ZjCYP and 13181 can be used for biosynthesis of 23-hydroxy dammarenediol and 25-hydroxy dammarenediol in a wild jujube saponin biosynthesis pathway.

Inventors

  • TONG XIAOLIN
  • TANG JUN
  • ZHAO LINHUA
  • LIU TAO

Assignees

  • 中国科学院天津工业生物技术研究所

Dates

Publication Date
20260508
Application Date
20241101

Claims (10)

  1. 1. The gene ZjCYP and 13181 of the wild jujube CYP450 enzyme is characterized in that the amino acid sequence of the coded protein is shown in SEQ ID NO. 2.
  2. 2. The wild jujube CYP450 enzyme gene ZjCYP13181,13181 of claim 1, wherein the nucleotide sequence is shown in SEQ ID No. 1.
  3. 3. The protein encoded by the wild jujube CYP450 enzyme gene ZjCYP and 13181 as claimed in claim 1 or 2, wherein the amino acid sequence of the encoded protein is shown in SEQ ID No. 2.
  4. 4. A recombinant plasmid comprising the wild jujube CYP450 enzyme gene ZjCYP13181,13181 of claim 1 or 2.
  5. 5. The recombinant plasmid according to claim 4, wherein the wild jujube CYP450 enzyme gene ZjCYP and the pESC-URA-PgDDS vector are subjected to homologous recombination to obtain the pESC-URA-PgDDS-ZjCYP13181 recombinant plasmid.
  6. 6. A genetically engineered bacterium comprising the recombinant plasmid of claim 4 or 5, or the exogenous wild jujube CYP450 enzyme gene ZjCYP and 13181 of claim 1 or 2 integrated into the genome of the genetically engineered bacterium.
  7. 7. The genetically engineered bacterium of claim 6, wherein the genetically engineered bacterium is a saccharomyces cerevisiae producing strain of which the starting bacterium is saccharomyces cerevisiae, more specifically 23-hydroxydammarenediol and/or 25-hydroxydammarenediol.
  8. 8. Use of the wild jujube CYP450 enzyme gene ZjCYP13181,13181 according to claim 1 or 2 for the preparation of wild jujube saponins, such as 23-hydroxydammarenediol and/or 25-hydroxydammarenediol.
  9. 9. The application of claim 8, wherein dammarenediol II is used as a substrate, and 23-hydroxy dammarenediol or 25-hydroxy dammarenediol is generated by hydroxylation of C-23 or C-25 of a side chain under the catalysis of a protein coded by the gene ZjCYP13181 of the wild jujube CYP450 enzyme; The dammarenediol II is synthesized by the ginseng dammarenediol synthase PgDDS on pESC-URA-PgDDS-ZjCYP13181 plasmid.
  10. 10. The use of the wild jujube CYP450 enzyme gene ZjCYP and 13181 according to claim 1 or 2 in cultivating wild jujube with high yield of wild jujube saponin, in particular, introducing the wild jujube CYP450 enzyme gene ZjCYP and 13181 into wild jujube to obtain wild jujube with high yield of wild jujube saponin, optionally further cultivating the wild jujube variety with high yield of wild jujube saponin.

Description

Wild jujube CYP450 enzyme gene ZjCYP13181 and application thereof in wild jujube saponin biosynthesis Technical Field The invention belongs to the technical field of biology, and particularly relates to a wild jujube CYP450 enzyme gene ZjCYP and 13181 and application thereof in biosynthesis of 23-hydroxy dammarenediol and 25-hydroxy dammarenediol in a synthetic pathway in wild jujube saponin biosynthesis. Background The semen Ziziphi Spinosae saponin A and B are active ingredients of traditional Chinese medicine semen Ziziphi Spinosae (semen Ziziphi Spinosae of Rhamnaceae). The wild jujube saponins A and B have the activities of treating insomnia, resisting anxiety, resisting insomnia, improving memory, treating Alzheimer disease and the like. The natural semen Ziziphi Spinosae contains low amounts of semen Ziziphi Spinosae saponin A and B. The spiny jujube seed saponin is produced by the traditional agricultural planting and extraction separation method, and has high production cost and long production period. The structure of the spine date seed saponin is complex, and the chemical synthesis is extremely difficult. Emerging synthetic biology brings a transition to efficient production of the wild jujube saponins, but this requires resolution of the biosynthetic pathway of the wild jujube saponins. Therefore, research on analysis of the biosynthesis path of the spine date seed saponin is carried out. The semen Ziziphi Spinosae saponin is used as a triterpene saponin, and comprises semen Ziziphi Spinosae sapogenin and glycosyl chain. From the biogenic route, the synthesis of the wild jujube sapogenin is the first and most critical step in the synthesis of the wild jujube sapogenin. However, no report has been made on the pathway of wild jujube sapogenin biosynthesis so far, and we have started the excavation of key enzymes in the pathway of wild jujube sapogenin biosynthesis from scratch. Disclosure of Invention The invention aims to provide a wild jujube CYP450 enzyme gene ZjCYP and 13181 which are involved in the biosynthesis of wild jujube saponins, and can be used for forming 23-hydroxy dammarenediol and 25-hydroxy dammarenediol in the biosynthesis of wild jujube sapogenin and for preparing the 23-hydroxy dammarenediol and the 25-hydroxy dammarenediol. In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: The first aspect of the invention provides a wild jujube CYP450 enzyme gene ZjCYP13181, the amino acid sequence of the coded protein is shown in SEQ ID NO.2, preferably the nucleotide sequence is shown in SEQ ID NO.1, and the total length of the sequence is 1554bp. The second aspect of the invention provides the protein coded by the gene ZjCYP and 13181 of the wild jujube CYP450 enzyme, the amino acid sequence of the coded protein is shown in SEQ ID NO.2, and 517 amino acid residues are coded. The third aspect of the present invention provides a recombinant plasmid containing the above-described wild jujube CYP450 enzyme gene ZjCYP 13181. Further, it is preferable that the wild jujube CYP450 enzyme gene ZjCYP13181 is subjected to homologous recombination with the pESC-URA-PgDDS plasmid to obtain a pESC-URA-PgDDS-ZjCYP13181 recombinant plasmid. The fourth aspect of the present invention provides a genetically engineered bacterium comprising the recombinant plasmid, or wherein the exogenous gene ZjCYP13181,13181 for the wild jujube CYP450 enzyme is integrated into the genome of the genetically engineered bacterium. Further, it is preferable that the transgenic engineering bacteria are Saccharomyces cerevisiae production strains of 23-hydroxydammarenediol and 25-hydroxydammarenediol The fifth aspect of the present invention provides an application of the wild jujube CYP450 enzyme gene ZjCYP13181 in preparing 23-hydroxy dammarenediol and 25-hydroxy dammarenediol. Further, the application of the wild jujube CYP450 enzyme gene ZjCYP13181 in preparing 23-hydroxy dammarenediol and 25-hydroxy dammarenediol is preferably characterized in that the wild jujube CYP450 enzyme gene ZjCYP13181 is catalyzed by the wild jujube CYP450 enzyme gene ZjCYP13181 and is subjected to side chain C-23 or C-25 hydroxylation to generate 23-hydroxy dammarenediol or 25-hydroxy dammarenediol by taking the dammarenediol II synthesized by the ginseng dammarenediol synthase PgDDS on the pESC-URA-PgDDS-ZjCYP13181 plasmid as a substrate. The sixth aspect of the present invention provides an application of the wild jujube CYP450 enzyme gene ZjCYP and 13181 in culturing wild jujube with high yield of wild jujube saponin, specifically, the wild jujube CYP450 enzyme gene ZjCYP and 13181 is introduced into wild jujube to obtain wild jujube with high yield of wild jujube saponin, optionally, further culturing the wild jujube variety with high yield of wild jujube saponin. The wild jujube CYP450 enzyme gene ZjCYP and 13181 are identified from wild jujube plants through transcriptome sequencing and bio