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CN-121991979-A - Wild jujube CYP450 enzyme gene ZjCYP and 30487 and application thereof

CN121991979ACN 121991979 ACN121991979 ACN 121991979ACN-121991979-A

Abstract

The invention relates to a wild jujube CYP450 enzyme gene ZjCYP and 30487 and application thereof in wild jujube saponin biosynthesis. The nucleotide sequence of the gene is shown as SEQ ID NO. 1, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 2. The wild jujube CYP450 enzyme gene ZjCYP and 30487 provided by the invention can be used for biosynthesis of 16-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 30487 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 ZjCYP30487,30,087 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 ZjCYP30487,30487 as claimed in claim 1 or 2, wherein the amino acid sequence of the encoded protein is shown as SEQ ID No. 2.
  4. 4. A recombinant plasmid comprising the wild jujube CYP450 enzyme gene ZjCYP30487,30487 of claim 1 or 2.
  5. 5. The recombinant plasmid according to claim 4, wherein the recombinant plasmid pESC-URA-PgDDS-ZjCYP30487 is obtained by homologous recombination of the wild jujube CYP450 enzyme gene ZjCYP30487,487 with the pESC-URA-PgDDS vector.
  6. 6. A genetically engineered bacterium comprising the recombinant plasmid of claim 4 or 5, or having integrated into its genome an exogenous wild jujube CYP450 enzyme gene ZjCYP30487,30487 of claim 1.
  7. 7. The genetically engineered bacterium of claim 6, wherein the genetically engineered bacterium is A strain of Saccharomyces cerevisiae producing 16-hydroxy dammarenediol.
  8. 8. Use of the wild jujube CYP450 enzyme gene ZjCYP30487,30487 according to claim 1 or 2 in the preparation of 16-hydroxy dammarenediol.
  9. 9. The application of claim 8, wherein dammarenediol II is used as a substrate, and 16-hydroxy dammarenediol is generated by hydroxylation of C-23 or C-25 of a side chain under the catalysis of a protein coded by ZjCYP and 30487 of the wild jujube CYP450 enzyme gene; the dammarenediol II is synthesized by the ginseng dammarenediol synthase PgDDS on the pESC-URA-PgDDS-ZjCYP30487 plasmid.
  10. 10. The use of the wild jujube CYP450 enzyme gene ZjCYP30487,30487 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 ZjCYP30487,30487 into wild jujube to obtain wild jujube with high yield of wild jujube saponin, optionally further cultivating wild jujube variety with high yield of wild jujube saponin.

Description

Wild jujube CYP450 enzyme gene ZjCYP and 30487 and application thereof Technical Field The invention belongs to the technical field of biology, and particularly relates to a wild jujube CYP450 enzyme gene ZjCYP and 30487 and application thereof in biosynthesis of 16-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 30487 which are involved in the biosynthesis of wild jujube saponins, and can be used for forming 16-hydroxy dammarenediol in the biosynthesis of wild jujube sapogenin and for preparing the 16-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 ZjCYP and 30487, the nucleotide sequence of the wild jujube CYP450 enzyme gene ZjCYP and 30487 is shown as SEQ ID NO.1, and the full length of the sequence is 1464bp. The second aspect of the invention provides the protein coded by the wild jujube CYP450 enzyme gene ZjCYP and 30487 amino acid residues, wherein the amino acid sequence of the coded protein is shown as SEQ ID NO. 2. The third aspect of the present invention provides a recombinant plasmid containing the above-described wild jujube CYP450 enzyme gene ZjCYP30487,30487. Further, it is preferable that the wild jujube CYP450 enzyme gene ZjCYP30487,087 is subjected to homologous recombination with the pESC-URA-PgDDS plasmid to obtain the pESC-URA-PgDDS-ZjCYP30487 recombinant plasmid. The fourth aspect of the present invention provides a genetically engineered bacterium WAT11, comprising the recombinant plasmid, or wherein the genome of the genetically engineered bacterium has the exogenous wild jujube CYP450 enzyme gene ZjCYP and 30487 integrated therein. Further, it is preferable that the transgenic engineering bacterium is a Saccharomyces cerevisiae production strain in which the transgenic engineering bacterium is 16-hydroxy dammarenediol The fifth aspect of the present invention provides an application of the wild jujube CYP450 enzyme gene ZjCYP30487,30487 in preparing 16-hydroxy dammarenediol. The dammarenediol II is taken as a substrate, and 16-hydroxy dammarenediol is generated by hydroxylation of a side chain at C-23 position or C-25 position under the catalysis of a protein coded by the wild jujube CYP450 enzyme gene ZjCYP and 30487; the dammarenediol II is synthesized by the ginseng dammarenediol synthase PgDDS on the pESC-URA-PgDDS-ZjCYP30487 plasmid. The invention obtains target protein after expression in recombinant bacteria through recombinant plasmid, and directly generates 16-hydroxy dammarenediol by further catalyzing substrate dammarenediol. The sixth aspect of the invention provides an application of the wild jujube CYP450 enzyme gene ZjCYP and 30487 in culturing wild jujube with high yield of wild jujube saponin, in particular to an application of introducing the wild jujube CYP450 enzyme gene ZjCYP and 30487 into wild jujube to obtain the wild jujube with high yield of wild jujube saponin, and optionally, further culturing the wild jujube variety with high yield of wild jujube saponin. The wild jujube CYP450 enzyme gene ZjCYP and 30487 are identified from wild jujube plants through transcriptome sequencing and bioinformatics, are screened after a large number of experiments, and are obtained through reverse transcription PC