Wild jujube CYP450 enzyme gene ZjCYP13181 and application of wild jujube CYP450 enzyme gene ZjCYP13181 in biological synthesis of jujuboside

Abstract

The invention relates to a wild jujube CYP450 enzyme gene ZjCYP13181 and an application of the wild jujube CYP450 enzyme gene ZjCYP13181 in biological synthesis of jujuboside. The nucleotide sequence of the gene is as shown in SEQ ID NO.1, and the full length of the sequence is 1554bp; the amino acid sequence of the encoded protein is as shown in SEQ ID NO.2, and 517 amino acid residues are encoded. The wild jujube CYP450 enzyme gene ZjCYP13181 disclosed by the invention can be used for biosynthesis of 23-hydroxyl dammar enediol and 25-hydroxyl dammar enediol in a biological synthesis path of jujuboside.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a jujube CYP450 enzyme gene ZjCYP13181 and its application in the biosynthesis of 23-hydroxydammarene diol and 25-hydroxydammarene diol in the biosynthesis pathway of jujube seed saponins. Background Technology

[0002] Jujube seed saponins A and B are the active ingredients of jujube seed (the kernel of the fruit of the jujube plant, which belongs to the Rhamnaceae family) in traditional Chinese medicine.

[0003] Jujube seed saponins A and B possess activities such as treating insomnia, anti-anxiety, improving memory, and treating Alzheimer's disease. The content of jujube seed saponins A and B in natural jujube seeds is low. Producing jujube seed saponins using traditional agricultural planting and extraction methods is costly and time-consuming. Jujube seed saponins have complex structures, making chemical synthesis extremely difficult. Emerging synthetic biology offers a breakthrough for the efficient production of jujube seed saponins, but this requires elucidating the biosynthetic pathways of jujube seed saponins. Therefore, we conducted research on the elucidation of the biosynthetic pathways of jujube seed saponins.

[0004] Jujube seed saponins, as triterpenoid saponins, are composed of jujube seed aglycone and glycosyl chains. From a biosynthetic perspective, the synthesis of jujube seed aglycone is the first and most crucial step in the synthesis of jujube seed saponins. However, there are currently no reports on the biosynthetic pathway of jujube seed aglycone. Therefore, we began our investigation from scratch to uncover the key enzymes involved in the biosynthetic pathway of jujube seed aglycone. Summary of the Invention

[0005] The purpose of this invention is to provide a jujube CYP450 enzyme gene ZjCYP13181 that participates in the biosynthesis of jujube seed saponins, which can be used to form 23-hydroxydammarene diol and 25-hydroxydammarene diol in the biosynthesis of jujube seed saponins, and can also be used to prepare 23-hydroxydammarene diol and 25-hydroxydammarene diol.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] The first aspect of the present invention provides a jujube CYP450 enzyme gene ZjCYP13181, the amino acid sequence of the protein encoded by which is shown in SEQ ID NO.2, preferably, the nucleotide sequence of which is shown in SEQ ID NO.1, with a full length of 1554 bp.

[0008] The second aspect of the present invention provides a protein encoded by the jujube CYP450 enzyme gene ZjCYP13181, the amino acid sequence of which is shown in SEQ ID NO.2, encoding 517 amino acid residues.

[0009] A third aspect of the present invention provides a recombinant plasmid containing the above-mentioned jujube CYP450 enzyme gene ZjCYP13181.

[0010] Furthermore, preferably, the jujube CYP450 enzyme gene ZjCYP13181 is homologously recombined with the pESC-URA-PgDDS plasmid to obtain the pESC-URA-PgDDS-ZjCYP13181 recombinant plasmid.

[0011] The fourth aspect of the present invention provides a transgenic engineered bacterium containing the above-mentioned recombinant plasmid, or wherein the genome of the genetically engineered bacterium is integrated with the exogenous jujube CYP450 enzyme gene ZjCYP13181.

[0012] Furthermore, preferably, the transgenic engineered bacteria is a *Saccharomyces cerevisiae* strain that produces 23-hydroxydammarene glycol and 25-hydroxydammarene glycol.

[0013] The fifth aspect of this invention provides the application of the above-mentioned jujube CYP450 enzyme gene ZjCYP13181 in the preparation of 23-hydroxydammarenediol and 25-hydroxydammarenediol.

[0014] Furthermore, preferably, the application of the jujube CYP450 enzyme gene ZjCYP13181 in the preparation of 23-hydroxydammarene diol and 25-hydroxydammarene diol is characterized by: using dammarene diol II synthesized by ginseng dammarene diol synthase PgDDS on the pESC-URA-PgDDS-ZjCYP13181 plasmid as a substrate, and then catalyzing the jujube CYP450 enzyme ZjCYP13181 encoded by the jujube CYP450 enzyme gene ZjCYP13181, the side chain is hydroxylated at the C-23 or C-25 position to generate 23-hydroxydammarene diol or 25-hydroxydammarene diol.

[0015] The sixth aspect of this invention provides the application of the jujube CYP450 enzyme gene ZjCYP13181 in the cultivation of jujubes with high yield of jujube seed saponins. Specifically, the jujube CYP450 enzyme gene ZjCYP13181 is introduced into jujubes to obtain jujubes with high yield of jujube seed saponins. Optionally, it can be further cultivated into a jujube variety with high yield of jujube seed saponins.

[0016] The Ziziphus jujuba CYP450 enzyme gene ZjCYP13181 described in this invention was identified from Ziziphus jujuba plants through transcriptome sequencing and bioinformatics techniques, and was obtained after extensive experimental screening. It was obtained through reverse transcription PCR using mRNA as a template and further sequence-specific primer PCR. The target protein was obtained by expressing the recombinant plasmid in Saccharomyces cerevisiae WAT11, and then further catalyzed by the substrate dammarene diol to directly generate 23-hydroxydammarene diol and 25-hydroxydammarene diol.

[0017] This invention provides the CYP450 enzyme gene ZjCYP13181, which can be used as a regulatory gene for the biosynthesis of 23-hydroxydammarenediol and 25-hydroxydammarenediol, and can be applied to the preparation of 23-hydroxydammarenediol and 25-hydroxydammarenediol.

[0018] (1) With the popularization of plant genome sequencing and transcriptome sequencing, the biosynthetic pathways of more and more plant active ingredients have been elucidated, greatly promoting the development of synthetic biology. In this invention, the biosynthetic gene of the upstream precursors 23-hydroxydammarenediol and 25-hydroxydammarenediol in the jujube seed saponin biosynthetic pathway, namely the CYP450 enzyme gene ZjCYP13181, was first identified and successfully verified in jujube, opening up a new method for the biosynthesis of the upstream precursors 23-hydroxydammarenediol and 25-hydroxydammarenediol in the jujube seed saponin biosynthetic pathway. This invention obtains the target product by heterologously expressing the ginseng-derived dammarenediol synthase PgDDS and the jujube CYP450 enzyme gene ZjCYP13181 in *Saccharomyces cerevisiae* WAT11, which has the advantages of single component and high yield.

[0019] (2) This invention provides a recombinant plasmid containing the CYP450 enzyme gene ZjCYP13181 and a genetically engineered bacterium, which lays the foundation for the large-scale synthesis of the upstream precursors 23-hydroxydammarenediol and 25-hydroxydammarenediol in the jujube seed saponin biosynthesis pathway through bioengineering methods, and further lays the foundation for the research on constructing cell factories that produce jujube seed saponins.

[0020] (3) By heterologous biosynthesis of 23-hydroxydammarene diol and 25-hydroxydammarene diol by Saccharomyces cerevisiae, the production cycle is short, the controllability is strong, the fermentation product is single, and it is easy to separate and purify in the later stage. It does not have the disadvantages of traditional agricultural planting and extraction separation methods, such as high production cost, long production cycle, and product quality being easily affected by natural and social factors.

[0021] The CYP450 enzyme gene ZjCYP13181 of jujube is a key gene for the biosynthesis of 23-hydroxydammarenediol and 25-hydroxydammarenediol, upstream precursors in the jujube seed saponin biosynthesis pathway. It can also be used for breeding research on jujubes that produce high yields of jujube seed saponins. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the derived synthetic pathways for 23-hydroxydammarenediol and 25-hydroxydammarenediol;

[0023] Figure 2 A schematic diagram illustrating the construction of the recombinant expression plasmid pESC-URA-PgDDS-ZjCYP13181;

[0024] Figure 3 This study investigated the catalytic activity of the CYP450 enzyme gene ZjCYP13181 from Ziziphus jujuba var. spinosa on the substrate dammarene glycol II in yeast using HPLC (i: HPLC analysis data of fermentation products from strain WAT11 / pESC-URA-PgDDS-ZjCYP13181; ii: HPLC analysis data of dammarene glycol II standard; iii: HPLC analysis data of fermentation products from strain WAT11 / pESC-URA-PgDDS. 1 represents 2,3-hydroxydammarene glycol, 2 represents 2,5-hydroxydammarene glycol, and 3 represents dammarene glycol II).

[0025] Figure 4 The LC-MS detection results show the characteristic peak ion spectrum of reaction product 1 ([C30H52O3–2H2O+H)). + = 425.3778, [C30H52O3+Na] + = 483.3809, [C30H52O3+K] + =499.3548).

[0026] Figure 5 The LC-MS results show the characteristic peak ion spectrum of reaction product 2 ([C30H52O3–2H2O+H)). + = 425.3778, [C30H52O3+Na] + = 483.3809, [C30H52O3+K] + =499.3548).

[0027] Figure 6 The NMR results are for reaction product 1. 13 C spectrum.

[0028] Figure 7 The NMR results are for reaction product 1. 1 H spectrum.

[0029] Figure 8 The results are NMR detection results, showing the HSQC spectrum of reaction product 1.

[0030] Figure 9The results are NMR detection results, showing the HMBC spectrum of reaction product 1.

[0031] Figure 10 The NMR results are for reaction product 2. 13 C spectrum

[0032] Figure 11 The NMR results are for reaction product 2. 1 H spectrum.

[0033] Figure 12 The results are NMR detection results, and the HSQC spectrum of reaction product 2 is shown.

[0034] Figure 13 The results are NMR detection results, showing the HMBC spectrum of reaction product 2. Detailed Implementation

[0035] definition

[0036] As used herein, the terms “active polypeptide”, “polypeptide of the present invention and its derivative polypeptides”, “enzyme of the present invention”, and “ZjCYP13181 of the present invention” all refer to the ZjCYP13181 (SEQ ID NO.2) polypeptide and its derivative polypeptides.

[0037] As used herein, "isolated polypeptide" means that the polypeptide is substantially free of other naturally occurring or associated proteins, lipids, carbohydrates, or other substances. Those skilled in the art can purify the polypeptide using standard protein purification techniques. A substantially pure polypeptide will produce a single master band on a non-reducing polyacrylamide gel. The purity of the polypeptide can also be further analyzed using its amino acid sequence.

[0038] The active polypeptides of the present invention can be recombinant polypeptides, natural polypeptides, or synthetic polypeptides. The polypeptides of the present invention can be naturally purified products, chemically synthesized products, or produced from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, plants) using recombinant technology.

[0039] The present invention also includes fragments, derivatives, and analogs of the said polypeptide. As used herein, the terms “fragment,” “derivative,” and “analyte” refer to a polypeptide that substantially retains the same biological function or activity as the said polypeptide.

[0040] The polypeptide fragments, derivatives, or analogs of the present invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituent groups in one or more amino acid residues; or (iii) polypeptides formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) polypeptides formed by fusing an additional amino acid sequence to the polypeptide sequence (e.g., a leader sequence or secretion sequence or a sequence used to purify the polypeptide or a proteogen sequence, or a fusion protein formed with an antigen IgG fragment). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0041] The polynucleotides of this invention can be in DNA or RNA form. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide can be identical to or a degenerate variant of the coding region sequence shown in SEQ ID NO:1.

[0042] The term "polynucleotide encoding a polypeptide" can refer to a polynucleotide that includes the polypeptide, or it can also include additional coding and / or non-coding sequences.

[0043] This invention also relates to variants of the aforementioned polynucleotides that encode polypeptides or fragments, analogs, and derivatives of polypeptides having the same amino acid sequence as those of this invention. These polynucleotide variants can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially alter the function of the polypeptide it encodes.

[0044] The present invention also relates to polynucleotides that hybridize with the above-described sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize with the polynucleotides described herein under stringent (or stringent) conditions.

[0045] This invention also relates to nucleic acid fragments that hybridize with the above-described sequences. As used herein, a "nucleic acid fragment" is at least 15 nucleotides long, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides or more. The nucleic acid fragment can be used in nucleic acid amplification techniques (such as PCR) to identify and / or isolate polynucleotides encoding the ZjCYP13181 protein.

[0046] The polypeptides and polynucleotides in this invention are preferably provided in isolated form and are more preferably purified to homogenization.

[0047] The full-length nucleotide sequences or fragments thereof of the present invention can generally be obtained by PCR amplification, recombination, or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed in the present invention, especially the open reading frame sequences, and the relevant sequences can be amplified using commercially available cDNA libraries or cDNA libraries prepared according to conventional methods known to those skilled in the art as templates. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified from each amplification in the correct order.

[0048] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0049] In addition, sequences can be synthesized artificially, especially when the fragment length is short. Typically, long sequences can be obtained by first synthesizing multiple small fragments and then joining them.

[0050] Currently, the DNA sequence encoding the protein of this invention (or a fragment thereof, or a derivative thereof) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of this invention through chemical synthesis.

[0051] The application of PCR technology to amplify DNA / RNA is preferred for obtaining the gene of the present invention. Especially when it is difficult to obtain full-length cDNA from a library, the RACE method (RACE-cDNA end amplification method) is preferred. Primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein and can be synthesized using conventional methods. The amplified DNA / RNA fragments can be separated and purified using conventional methods such as gel electrophoresis.

[0052] The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well-known in the art. Any plasmid and vector can be used as long as it can replicate and remain stable within the host. An important characteristic of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and translational control elements.

[0053] Methods well known to those skilled in the art can be used to construct expression vectors containing the ZjCYP13181 polypeptide, along with the encoding DNA sequence and suitable transcription / translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, and in vivo recombination techniques. The DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters include: the lac or trp promoter of *E. coli*; the PL promoter of *λ* phage; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, early and late SV40 promoters, retroviral LTRs, and other known promoters that control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0054] In addition, the expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting host cells for transformation, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0055] Vectors containing the appropriate DNA sequence and appropriate promoter or control sequence can be used to transform appropriate host cells so that they can express proteins.

[0056] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; Salmonella typhimurium bacterial cells; fungal cells such as yeast; plant cells; Drosophila S2 or Sf9 insect cells; and animal cells such as CHO, COS, 293 cells, or Bowes melanoma cells.

[0057] When the polynucleotides of this invention are expressed in higher eukaryotic cells, the insertion of an enhancer sequence into the vector will enhance transcription. Enhancers are cis-acting factors of DNA, typically approximately 10 to 300 base pairs, that act on the promoter to enhance gene transcription. Examples include the SV40 enhancer (100 to 270 base pairs) located late on the replication origin side, the polyoma enhancer located late on the replication origin side, and adenovirus enhancers.

[0058] Those skilled in the art are well aware of how to select appropriate vectors, promoters, enhancers, and host cells.

[0059] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as *E. coli*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0060] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0061] The recombinant peptides used in the methods described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0062] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0063] Experimental methods in the following examples, unless otherwise specified, were performed under standard conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0064] Materials and reagents

[0065] YPD medium: 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose. If preparing a solid medium, add 20 g / L agar powder.

[0066] SC-ΔUra medium: 6.7 g / L amino-free yeast nitrogen source, 100 mg / L Lys, 100 mg / L His, 100 mg / L LEu, 20 g / L glucose, 2 g / L SC Dropoutmix-ΔUra, Lys, His, Leu;

[0067] The expression vector pESC-URA-PgDDS for the Saccharomyces cerevisiae WAT11 was a plasmid previously constructed in the laboratory for the production of dammarene diol II.

[0068] Ginseng dammarene diol synthase PgDDS was synthesized by Genewiz Biotechnology Co., Ltd.

[0069] Dammarene diol II was purchased from Yunnan Xili Biotechnology Co., Ltd.; the PrimeScript reverse transcription kit was also used. TM The RT reagent kit was purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd.; the high-fidelity enzyme PhantaMax Super-Fidelity DNA Polymerase was purchased from Thermo Fisher Scientific (China) Co., Ltd.; and the homologous recombinase ClonExpress II One Step Cloning Kit was purchased from Nanjing Novizan Biotechnology Co., Ltd.

[0070] Example

[0071] Based on the functional annotation information of the jujube transcriptome, candidate CYP450 genes were screened from the sequencing annotation results. Finally, 30 CYP450 oxidase genes that are likely related to triterpenoid oxidation and have relatively high expression levels were selected. Among them, ZjCYP13181 was functionally annotated as a CYP450 monooxygenase. The nucleotide sequence of the Unigene was extracted from the FASTA file according to the Unigene ID (LOC107413181) for subsequent analysis.

[0072] Template cDNA was obtained by reverse transcription of mRNA using RT-PCR, and the ZjCYP13181 gene fragment was obtained by sequence-specific primer PCR. This fragment was then constructed into pESC-URA-PgDDS, and the plasmid was subsequently introduced into the WAT11 engineered strain for fermentation. Following a series of procedures including HPLC, LC-MS detection, and NMR identification, it was finally determined that the ZjCYP13181 gene can catalyze the hydroxylation of the dammarene diol II side chain at position 23 or 25 to generate 23-hydroxydammarene diol and 25-hydroxydammarene diol (…). Figure 1 ).

[0073] The steps for each stage of heterologous expression of ZjCYP13181 are as follows:

[0074] (1) Obtaining the ZjCYP13181 gene

[0075] Nucleotide sequences of 30 candidate genes were obtained from the FASTA file, and corresponding primers were designed based on the ORF. The reverse transcription was performed using the PrimeScript reverse transcription kit. TM The RT reagent kit was used to reverse transcribe the mRNA from jujube to obtain cDNA. Using the jujube cDNA as a template, PCR was performed with specific primers to obtain candidate genes. The specific sequence of ZjCYP13181 is ZjCYP13181-F: ggcggccgcactagtatcgaATGGAGGTTCCAGAGCTTGAG; (SEQ ID NO.3)

[0076] ZJCYP13181-R: agaattgttaattaagagctcTTAAAGCTTCCTCATTATCAAGTCCA. (SEQ IDNO.4)

[0077] (2) Construction of gene heterologous expression vectors

[0078] See the schematic diagram of the gene heterologous expression vector. Figure 2 First, the vector pESC-URA-PgDDS was linearized, and the linearized vector was obtained by double digestion with SacI and ClaI. The candidate gene fragment was ligated to the linearized vector using the ClonExpress II One Step Cloning Kit. After the reaction, the product was transformed into DH5α(DE3) competent cells and plated on LB agar plates containing ampicillin. Positive single colonies were picked the next day for colony PCR verification. Plasmids from PCR-positive strains were extracted and sent to a sequencing company for sequencing verification. Finally, a heterologous gene expression vector was obtained.

[0079] (3) Heterologous expression of Saccharomyces cerevisiae WAT11 / pESC-URA-PgDDS-ZjCYP13181

[0080] The constructed heterologous expression vector pESC-URA-PgDDS-ZjCYP13181 and the empty pESC-URA-PgDDS plasmid were transformed into the WAT11 strain using lithium acetate conversion to obtain a heterologous expression strain. The heterologous expression strain was then fermented, with the WAT11 strain containing only pESC-URA-PgDDS serving as a control group. The fermentation method was as follows: a single colony was picked from an SC-ΔURA plate and inoculated into 5 mL of SC-ΔURA liquid medium for overnight culture; the next day, 1 mL of bacterial culture was inoculated into 50 mL of SC-ΔURA (2% glucose) liquid medium and cultured at 30°C and 200 rpm for 24 hours; then, the bacterial culture was centrifuged at 3000 rpm for 3 minutes, the supernatant was discarded, and bacterial cells were obtained; the bacterial cells were washed once with 30 mL of sterile water and then added to 50 mL of SC-ΔURA...

[0081] Continue culturing (2% galactose) at 30°C and 200 rpm for 48 hours.

[0082] (4) Detection of heterologous expression products

[0083] After fermentation, the yeast cells were collected by centrifugation. 20 mL of lysis buffer (20% KOH, 50% EtOH) was added to the cells and the mixture was sonicated for 2 hours. Then, an equal volume of n-hexane was added for extraction twice. The extract was evaporated using a rotary evaporator to remove the n-hexane, yielding a crude extract. 1 mL of methanol was added to the crude extract, and the mixture was centrifuged at 12000 rpm for 10 minutes before being analyzed by HPLC.

[0084] The HPLC detection conditions are as follows:

[0085] The instrument used for HPLC analysis was a Shimadzu high-performance liquid chromatograph. The chromatographic column was a SilGreen column (250*4.6mm I.DS-5μm, 12nm), and the column temperature was 40℃. The mobile phase was water (A)-acetonitrile (B), with gradient elution: 0–20 min, 5%–100% B; 20–28 min, 100% B; 28–33 min, 5% B.

[0086] Injection volume: 20 μL; Flow rate: 1 mL / min; Detection wavelength: 190 nm. Detection results are shown below. Figure 3 This indicates that the heterologous expression strain WAT11 / pESC-URA-PgDDS-ZjCYP13181 produces new products 1 and 2 during heterologous expression.

[0087] LC-MS analysis of the fermentation products of WAT11 / pESC-URA-PgDDS-ZjCYP13181 revealed that the molecular weights of the newly appearing peaks 1 and 2 were consistent with the molecular weights of the products after monohydroxylation of dammarene diol II. Figure 4 , Figure 5).

[0088] (5) Identification of compound structure

[0089] To determine the structures of 1 and 2, 1 L of WAT11 / pESC-URA-PgDDS-ZjCYP13181 was fermented using the method described above. Cells were collected using the same method, and crude extracts were obtained. 1 and 2 were then prepared using semi-preparative HPLC. NMR spectroscopy (C, H, HSQC, and HMBC data for 1 and 2 were collected.) Figure 6-13 Based on previously reported NMR data of dammarene diol II, 1 and 2 were determined to be dammarene diol II with hydroxylation at C-23 and C-25 positions, respectively.

Claims

1. A CYP450 enzyme gene of jujube. ZjCYP13181 Its characteristics are, The amino acid sequence of the protein it encodes is shown in SEQ ID NO.

2.

2. The jujube CYP450 enzyme gene as described in claim 1 ZjCYP13181 Its characteristics are, Its nucleotide sequence is shown in SEQ ID NO.

1.

3. The jujube CYP450 enzyme gene according to claim 1 or 2 ZjCYP13181 The encoded protein is characterized by, The amino acid sequence of the encoded protein is shown in SEQ ID NO.

2.

4. Contains the jujube CYP450 enzyme gene as described in claim 1 or 2. ZjCYP13181 The recombinant plasmid.

5. The recombinant plasmid according to claim 4, characterized in that, The CYP450 enzyme gene of jujube ZjCYP13181 Homologous recombination with the pESC-URA-PgDDS vector yielded the recombinant plasmid pESC-URA-PgDDS-ZjCYP13181.

6. A genetically engineered bacterium, characterized in that, It contains the recombinant plasmid as described in claim 4 or 5, or the genome of the genetically engineered bacterium is integrated with an exogenous jujube CYP450 enzyme gene as described in claim 1 or 2. ZjCYP13181 .

7. The genetically engineered bacterium according to claim 6, characterized in that, The genetically engineered strain is a *Saccharomyces cerevisiae* strain, more specifically a *Saccharomyces cerevisiae* production strain of 23-hydroxydammarene glycol and / or 25-hydroxydammarene glycol.

8. The jujube CYP450 enzyme gene according to claim 1 or 2 ZjCYP13181 Application in the preparation of jujube seed saponins, such as 23-hydroxydammarenediol and / or 25-hydroxydammarenediol.

9. The application according to claim 8, characterized in that: Using dammarene diol II as a substrate, the CYP450 enzyme gene of jujube was described above. ZjCYP13181 The encoded protein undergoes hydroxylation at the C-23 or C-25 position of its side chain to generate 23-hydroxydammarene glycol or 25-hydroxydammarene glycol under catalysis. The dammarene diol II is derived from ginseng dammarene diol synthase on the pESC-URA-PgDDS-ZjCYP13181 plasmid. PgDDS synthesis.

10. The jujube CYP450 enzyme gene according to claim 1 or 2 ZjCYP13181 In the cultivation of jujube seed saponins with high yield, specifically, the jujube CYP450 enzyme gene is used... ZjCYP13181 The jujube seeds were introduced into jujubes to obtain jujubes with high yields of jujube seed saponins. Selectively, these jujubes were further cultivated into jujube varieties with high yields of jujube seed saponins.

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