CN117327715B - Bacopa monnieri P450 enzyme gene BmCYP068 and application thereof - Google Patents

Abstract

The invention relates to a Bacopa monnieri P450 enzyme gene BmCYP and application thereof, belonging to the technical field of biology. The nucleotide sequence of the Bacopa monnieri P450 enzyme gene BmCYP and 068 is shown as SEQ ID NO.1, and the full 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 Bacopa monnieri P450 enzyme gene BmCYP and 068 can be used as biosynthesis regulatory genes of 23-OH-Dammarenediol II and 25-OH-Dammarenediol II, and can be applied to preparation of the genes, so that the application prospect is obvious and the popularization and application are easy.

Description

A Bacopa monnieri P450 enzyme gene BmCYP068 and its application

Technical Field

The invention belongs to the field of biotechnology, and specifically relates to a Bacopa monnieri P450 enzyme gene BmCYP068 and an application thereof.

Background technique

Bacopa monnieri (L.) Wettst. is a plant of the genus Bacopa Aubl. in the family Scrophulariaceae. It is mainly distributed in tropical and subtropical regions, and is often found in places such as watersides, wetlands and beaches. In my country, it is mainly distributed in Taiwan, Fujian, Guangdong, Yunnan and other provinces. Bacopa monnieri is a highly respected herb in traditional Indian Ayurvedic medicine. It is recognized as a high-value medicinal plant for brain tonic, which can improve memory and intelligence. Different research groups around the world are exploring the use of Bacopa monnieri for the treatment of dementia, amnesia, memory dysfunction, Parkinson's disease, Alzheimer's disease, epileptic seizures and schizophrenia. In addition to neuroprotection, Bacopa monnieri also has sedative, antibacterial, anti-inflammatory, anticonvulsant, anti-aging, bronchial vasodilator, anticancer, antidepressant, antiemetic and anti-ulcer activities. It is reported that these potential pharmacological activities of Bacopa monnieri are closely related to its secondary metabolites, dammarane-type triterpenoid saponins.

The triterpenoid saponins in Bacopa monnieri are rich in content and of various types. According to their structure, they can be divided into dammarane type, cucurbitane type, lupeol type, ursane type, etc. Among them, dammarane type triterpenoid saponins are the most common type of components, and these compounds are closely related to the potential pharmacological effects of plants. This type of compound mostly exists in the form of glycosides, and its parent core structure is mainly jujube sapogenin and pseudojujube sapogenin. The difference between these compounds lies in the variety of types and methods of connected sugar groups.

The triterpenoid saponins in Bacopa monnieri are low in content and complex in structure, and a large amount of raw materials are required to extract them from plant materials. However, most of the existing resources of Bacopa monnieri are in the wild state, with a small number and scattered distribution, and the cost of separation is high and the chemical synthesis is difficult, which limits the application of triterpenoid saponins in Bacopa monnieri. Therefore, using synthetic biology methods to produce monomer compounds is one of the most effective and feasible methods to achieve large-scale production of triterpenoid saponins in Bacopa monnieri in the future. However, the current research on Bacopa monnieri mainly focuses on the extraction and separation of compounds and the identification of pharmacological activities, and the molecular mechanism of the synthesis of triterpenoid saponins is still unclear. In order to obtain dammarane-type triterpenoid saponins in Bacopa monnieri by biosynthesis, it is first necessary to analyze the biosynthetic pathway of these compounds.

Jujubosapogenin and pseudojujubosapogenin are key intermediates of triterpenoid saponins in Bacopa monnieri, and identification of their biosynthetic enzymes has become a top priority. However, to date, the functions of the enzymes responsible for the production of jujubosapogenin and pseudojujubosapogenin in Bacopa monnieri have not been verified, which has affected the advancement of the biosynthesis of dammarane-type triterpenoid saponins in Bacopa monnieri.

Summary of the invention

The purpose of the present invention is to solve the deficiencies of the prior art and to provide a Bacopa monnieri P450 enzyme gene BmCYP068, which can be used as a regulatory gene for forming 23-OH-Dammarenediol II and 25-OH-Dammarenediol II in the biosynthesis of Bacopa monnieri triterpenoid saponins and for preparing 23-OH-Dammarenediol II and 25-OH-Dammarenediol II.

To achieve the above purpose, the technical solution adopted by the present invention is as follows:

In a first aspect, the present invention provides a Bacopa monnieri P450 enzyme gene BmCYP068. The nucleotide sequence of the Bacopa monnieri P450 enzyme gene BmCYP068 is shown in SEQ ID NO.1, and the full length of the sequence is 1554 bp.

The second aspect of the present invention provides a protein encoded by the Bacopa monnieri P450 enzyme gene BmCYP068. The amino acid sequence of the encoded protein is shown in SEQ ID NO. 2, encoding 517 amino acid residues.

The third aspect of the present invention provides a recombinant plasmid containing the Bacopa monnieri P450 enzyme gene BmCYP068.

Furthermore, preferably, the Bacopa monnieri P450 enzyme gene BmCYP068 is homologously recombined with the Y33 vector to obtain the Y33-BmCYP068 recombinant plasmid.

The fourth aspect of the present invention provides a genetically modified engineered bacterium, comprising the above-mentioned recombinant plasmid, or the genome of the genetically modified bacterium is integrated with the above-mentioned exogenous Bacopa monnieri P450 enzyme gene BmCYP068.

Furthermore, preferably, the genetically modified engineered bacteria are Saccharomyces cerevisiae Dammarenediol II chassis cells.

The fifth aspect of the present invention provides the Bacopa monnieri P450 enzyme BmCYP068 encoded by the Bacopa monnieri P450 enzyme gene BmCYP068.

The sixth aspect of the present invention provides the use of the Bacopa monnieri P450 enzyme gene BmCYP068 in the preparation of 23-OH-Dammarenediol II and 25-OH-Dammarenediol II.

Furthermore, preferably, Dammarenediol II produced by chassis cells is used as a substrate, and is hydroxylated at position 23 or 25 of the side chain under the catalysis of the Bacopa P450 enzyme BmCYP068 encoded by the Bacopa P450 enzyme gene BmCYP068 to generate 23-OH-Dammarenediol II and 25-OH-Dammarenediol II.

The invention obtains the target protein after expressing the target protein in the chassis cells of Saccharomyces cerevisiae through the recombinant plasmid, and directly generates 23-OH-Dammarenediol II and 25-OH-Dammarenediol II by further catalyzing the substrate Dammarenediol II.

The Bacopa monnieri P450 enzyme gene BmCYP068 of the present invention is identified from Bacopa monnieri plants through transcriptome sequencing and bioinformatics technology, and screened after a large number of experiments; it is obtained through codon optimization and artificial synthesis. The amplification primers of the Bacopa monnieri P450 enzyme gene BmCYP068 are as follows:

5'F: ATGGCAGTTAAATTGAGCAGC; (SEQ ID NO.3)

3'R:TTACAATTTGTGCAAAACCATAGAAGC. (SEQ ID NO.4)

In addition, when homologous recombination is performed with vector Y33, the BmCYP068 gene needs to be amplified and recovered using primers with homologous walls. The primers with homologous walls are as follows:

Upstream homology arm primer: 5'F: cagtcgacctcgaatctaga ATGGCAGTTAAATTGAGCAGC; (SEQ ID NO. 5).

Downstream homology arm primer: 3'R: ctaattacatgatgcggcccTTACAATTTGTGCAAAACCATAGAAGC (SEQ ID NO. 6).

The P450 enzyme gene BmCYP068 isolated and identified from Bacopa monnieri can be used as an important marker gene for molecular-assisted breeding of Bacopa monnieri, and can also be used as an important candidate gene for the production of 23-OH-Dammarenediol II, 25-OH-Dammarenediol II and Bacopa saponins in the construction of yeast chassis cells.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention provides a P450 enzyme gene BmCYP068 which can be used as a biosynthesis regulatory gene for 23-OH-Dammarenediol II and 25-OH-Dammarenediol II and is used in the preparation of

23-OH-Dammarenediol II and 25-OH-Dammarenediol II.

(1) With the rapid development of bioinformatics technology, the mining of key enzyme genes in the biosynthesis pathway of triterpenoid compounds has been greatly promoted. The biosynthesis regulatory gene of 23-OH-Dammarenediol II and 25-OH-Dammarenediol II in the present invention, namely the P450 enzyme gene BmCYP068, was first identified and successfully verified in Bacopa monnieri, opening up a new biosynthetic method for producing 23-OH-Dammarenediol II and 25-OH-Dammarenediol II. The present invention obtains the target product by heterologously expressing the protein in Saccharomyces cerevisiae and catalyzing it, adopts in vivo biosynthesis, and performs directional production, which has the advantages of high yield, etc.

(2) The present invention provides a recombinant plasmid and a genetically engineered bacterium containing the P450 enzyme gene BmCYP068, which lays a foundation for synthesizing 23-OH-Dammarenediol II and 25-OH-Dammarenediol II in large quantities through bioengineering methods, and further for constructing a cell factory that produces 23-OH-Dammarenediol II and 25-OH-Dammarenediol II.

(3) The heterologous biosynthesis of 23-OH-Dammarenediol II and 25-OH-Dammarenediol II has strong controllability, can reduce the demand for raw material planting, produce high product yield, and facilitate the subsequent separation and purification of 23-OH-Dammarenediol II and 25-OH-Dammarenediol II; it can also reduce the difficulties of chemical synthesis and the complexity of the synthesis path. The P450 enzyme gene BmCYP068, as a key gene for the biosynthesis of 23-OH-Dammarenediol II and 25-OH-Dammarenediol II, can also be used in the breeding research of Bacopa monnieri plants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic diagram of the synthetic pathway derived from 23-OH-Dammarenediol II and 25-OH-Dammarenediol II;

FIG2 is a schematic diagram of the construction of the recombinant expression plasmid Y33-BmCYP068;

Figure 3 is the electrophoresis detection result of the recombinant Bacopa monnieri P450 enzyme gene BmCYP068, where M is the nucleic acid Mar, and 1-6 are the positive single colony detection results;

Figure 4 is a HPLC detection of the catalytic effect of Bacopa monnieri P450 enzyme gene BmCYP068 on the substrate Dammarenediol II in yeast (DM+PPD standard: peak time of Dammarenediol II standard and PPD standard; DM chassis+Y33 empty load: the reaction result of the control group Y33 empty plasmid as a positive control; DM chassis+BmCYP068: the reaction result of Bacopa monnieri P450 enzyme gene BmCYP068 catalyzing the substrate Dammarenediol II in yeast, peak time of products 1 and 2).

FIG5 is the LC-MS detection result, the characteristic peak ion diagram of the reaction product 1 (theoretical molecular weight 460).

FIG6 is the LC-MS detection result, the characteristic peak ion diagram of the reaction product 2 (theoretical molecular weight 460).

FIG. 7 is the NMR detection result, NMR ¹³ C spectrum of the reaction product 1. FIG.

FIG8 is the NMR detection result, NMR ¹ H spectrum of the reaction product 1. ...

FIG. 9 is the NMR detection result, 1HMBC spectrum of the reaction product.

FIG. 10 is the NMR detection result and the HSQC spectrum of the reaction product 1.

Figure 11 shows the NMR detection results, COSY spectrum of reaction product 1

FIG. 12 is the NMR detection result, ROESY spectrum of reaction product 1.

FIG. 13 is the NMR detection result, NMR ¹³ C spectrum of the reaction product 2.

FIG. 14 is the NMR detection result, NMR ¹ H spectrum of the reaction product 2. FIG.

FIG. 15 is the NMR detection result, HMBC spectrum of the reaction product 2.

FIG. 16 is the NMR detection result, HSQC spectrum of the reaction product 2.

Figure 17 shows the NMR detection results, COSY spectrum of reaction product 2

FIG. 18 is the NMR detection result, ROESY spectrum of the reaction product 2.

Detailed ways

The present invention is further described in detail below in conjunction with embodiments.

Those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the art or the product specifications are used. If the manufacturer of the materials or equipment used is not specified, they are all conventional products that can be purchased.

Example 1

Based on the basic functional annotation information of the Unigene transcriptome of Bacopa monnieri, CYP candidate genes were screened in the sequencing annotation results. At the same time, the cytochrome P450 monooxygenase (CYPs) identified in plants was used as the reference sequence. The local BLAST analysis of the sequence was performed, and then the screening results were sorted and analyzed. Finally, 20 CYP genes were screened. Among them, the functional annotation of BmCYP068 was cytochrome P450 monooxygenase. Finally, according to the ID number corresponding to Unigene, the Unigene nucleotide sequence was extracted from the fasta file for subsequent analysis. After a series of work such as codon optimization, artificial synthesis, homologous recombination, in vivo induced expression in yeast chassis cells, yeast metabolite extraction, and HPLC, LC-MS and NMR detection, the BmCYP068 gene was finally identified as being able to catalyze the hydroxylation of the 23- or 25-position of the side chain of Dammarenediol II to generate 23-OH-Dammarenediol II and 25-OH-Dammarenediol II (Figure 1). The steps of each stage of the synthesis of 23-OH-Dammarenediol II and 25-OH-Dammarenediol II are as follows (the reagents, raw materials, instruments and equipment used in the following implementation are all commercially available):

(1) Codon optimization and artificial synthesis of BmCYP068 gene

The P450 synthase encoding gene BmCYP068 annotated from the transcriptome was codon-optimized and then artificially synthesized by the company.

(2) Construction and identification of gene recombination vectors

The schematic diagram of homologous recombination is shown in Figure 2. First, the vector Y33 was linearized, and the linearized vector was obtained by single restriction digestion with XbaI enzyme. The restriction digestion system (50μL) was 1μg of circular Y33 vector, 5μL of 10×NEBuffer, 1μL of RestrictionEnzyme, and double distilled water was added to the total system of 50μL. The reaction solution was placed at 37℃ for 15min to obtain the linearized vector Y33. The EasyPure Quick Gel Extraction Kit was used for recovery, and its concentration was measured after recovery. Finally, it was stored in a -20℃ refrigerator for standby use. During homologous recombination, the assembly was carried out according to the operating instructions of the homologous recombination enzyme kit, and then the amount of each component was calculated according to the concentration of the insert and the vector and the recombination instructions; finally, each component was added to the PCR reaction tube on ice, as shown in Table 1. The recombinant plasmid was obtained by homologous recombination of the Bacopa monnieri P450 enzyme gene BmCYP068 with the Y33 vector, named Y33-BmCYP068. The recombinant plasmid was transformed into DH5α E. coli, and the positive clones were selected for detection and sent to the company for sequencing. The electrophoresis detection results after assembly are shown in Figure 3, indicating that the assembly was successful. The recombinant operation was performed according to the following process:

Table 1 Candidate gene recombination reaction system

Table 1 Candidate genes Recombination System

Where X = (0.02 × Y33 base pairs) ng/linearized Y33 concentration ng/μL; Y = (0.02 × Y33 base pairs) ng/BmCYP068 recovery concentration ng/μL;

(3) Recombinant plasmid extraction

10 μL of the positive monoclonal stock solution detected by sequencing was placed in 6 mL LB liquid medium (100 mg/mL Amp) and cultured overnight at 37°C in a shaker (220 r/min). The plasmid was extracted according to the instructions in the plasmid DNA mini-preparation kit centrifugal column type (GenStar, Shenzhen, China). After extraction, the recovery concentration of the plasmid DNA was measured on a NanoReady ultra-micro UV-visible spectrophotometer, and finally stored in a -20°C refrigerator for later use.

(4) Preparation of competent DM yeast cells

Pick the DM-based cell strain on the SC-His-Leu plate and inoculate it into 100ml of SC-His-Leu liquid medium. Cultivate at 30℃ and 220rpm until OD=0.8-0.9. Use a 50mL centrifuge tube to separate and collect the bacteria at 5000g/5min. Wash twice with 25mL sterile water, repeat the centrifugation step, resuspend the washed bacteria with 1mLddH2o, transfer to a 1.5mL centrifuge tube, centrifuge at 13000rpm for 30s to collect the bacteria. Resuspend with 600μL dd water and dispense 100μL into each tube for transformation.

(5) DM yeast cell transformation

Centrifuge for 20 seconds in a handheld centrifuge, discard the supernatant, add the transformation system: PEG4000 (50%) 240μL, LAC (lithium acetate) 1.0mol 36μL, SSDNA (frog fish sperm) 2.0ug/μL 10μL, plasmid with target fragment (400ng) and ddH2O 74μL in total. Resuspend the mixed body and keep it at 30℃ for 20min, heat shock at 42℃ for 40min, take 200μL of bacterial solution to plate SC-His-Leu-Ura, and invert it into a 30℃ incubator for 2-4 days, then select the positive clone strain for verification.

(6) Screening of positive strain clones

Add 20 μL ddH2O to each 0.2mL PCR tube, pick 4 single colonies from the above culture; react at 95℃ for 10min in a PCR instrument for cell wall breaking. Add the following reaction system to the PCR tube: 12.5μL Super 2x Mix, 10.5μL sterilized water, 0.5μL universal forward primer (10mM), 0.5μL universal reverse primer (10mM), 1μL monoclonal template dissolved in water. Among them, the universal primer for detection was designed by software (SnapGene 3.2.1), the upstream primer for detection: GATGCTTTCTTTTTCTTTTTTTTTACAGATC, the downstream primer: GCGTGAATGTAAGCGTGAC; the PCR amplification cycle parameters are: 95℃3min; 95℃30s, 55℃30s, 72℃90S, 35 cycles; 72℃5min, 10℃5min.

(7) Detection and sequencing

Take 1.0μL Loading buffer and add 5μL of the above PCR product, mix well, and detect the amplification results by 1% agarose gel electrophoresis. If the size is similar to the target fragment, it is a true positive yeast strain, indicating successful transformation. The bacterial solution identified as positive clones was cultured in SC-His-Leu-Ura liquid medium for 1 day and then preserved. The method is to add 50% glycerol and bacterial solution in a 1:1 ratio to the seed preservation tube, mix well, and store in a -80℃ ultra-low temperature refrigerator for use.

(8) Yeast induced expression and detection

The positive transformed strains with normal growth were selected and cultured in SC-His-Leu-Ura liquid medium (50 ml). After culturing at 30°C and 220 rpm for 5 days, the cells were collected by centrifugation at 8000 rpm for 5 min, and the cells were lysed and broken with 2 ml of lysis solution (20% KOH, 50% EtOH); the cells were extracted three times with the same volume of n-hexane; the extract was dried by rotary evaporation and dissolved with 3 ml of chromatographic methanol. The extracted metabolites were analyzed by HPLC.

HPLC detection conditions are as follows:

The instrument used for HPLC detection is Agilent high performance liquid chromatograph. The chromatographic column is Agilent EC-C18 chromatographic column (4.6×100mm, 2.7um), column temperature: 25°C; the mobile phase for determination is: water (A)-acetonitrile (B), gradient elution: 0-10min, 70%-75% B; 10-20min, 75%-85% B; 20-22min, 85%-100% B; 22-25min, 100% B; the mobile phase A+B used during elution is 100% in total; linear gradient elution is used; elution time: 25min; injection volume: 10μL; flow rate: 0.8ml/min; detection wavelength is 203nm, and the detector is a diode array detector. The detection results are shown in Figure 5, indicating that the experimental sample has new products produced under the catalysis of Bacopa monnieri P450 enzyme BmCYP068.

LC-MS detection conditions are as follows:

In order to further confirm the reaction products detected by HPLC, Agilent 1290UPLC/6540Q-TOF liquid chromatography-mass spectrometry (LC/MS/MS) was used for detection: Mass spectrometry conditions: the ion source used was negative ion mode, voltage: 3500 V; fragmentation voltage: 135 V; cone voltage: 60 V; radio frequency voltage: 750 V, scanning range: 100-1000 m/z, scanning mode: SRM. Chromatographic conditions: The chromatographic column is an Agilent EC-C18 column (4.6×100mm, 2.7um), the column temperature is 25°C; the mobile phase is water (A)-acetonitrile (B), gradient elution: 0-10min, 70%-75% B; 10-20min, 75%-85% B; 20-22min, 85%-100% B; 22-25min, 100% B; the mobile phase A+B used in elution is 100% in total; linear gradient elution is used; elution time: 25min; injection volume: 10μL; flow rate: 0.8ml/min; detection wavelength is 203nm, and the detector is a diode array detector. MassHunter workstation (Agilent) software is used for data analysis. The test results are shown in Figures 5-6.

According to the above detection and analysis, it is speculated that DammarenediolII was hydroxylated at two sites under the catalysis of Bacopa monnieri P450 enzyme BmCYP068. In order to clarify the hydroxylation site, we performed NMR detection on compounds 1 and 2. NMR detection was performed on a Bruker AV-800MHz spectrometer (Santa Clara, USA), and the detection results are shown in Figures 7 to 18. Based on the above detection results, compound 1 was determined to be 23-OH-Dammarenediol II, and compound 2 was determined to be 25-OH-Dammarenediol II.

Sequence Listing

SEQ ID NO.1

Target sequence:

ATGGCTGTGAAACTAAGCTCAATCCTTATTTCTCTTCTCGTACTTGCACTCACAACATTGGTACTAAAAGTATTGAATTGGGTTTGGTTTAGACCCAAGAAATTGGAGAAAATCTTGAGAGATCAAGGCCTCAATGGAAACCCATACAGAATTCTTATTGGAGACATGAAGGACTTGATGTCTGCAACCAACGCAGAAAAATCCAGACCAATCCAACTTTCTGACGATATATCTCAGCATATGTTTCCGTATTACCATCAAGCCAGGAGCAAGTATGGACCGAATTCTTTTGTGTGGTTTGGACCCTCGCCAAAATTGATGATTGCTGATCCAGTTCTTCTCAAGGAGATATTCACAAAACCAGATGTATTTCATAAGCCTGTTCCAG ATCCCATTGGCCAATCCATTGCTGGAGGGCTTACGTTTCTTGAAGATGAAAAATGGGCCAAACACCGGAGAATTATTAATCCGGCTTTCCATATGGAGAAGCTCAAGGATAAGACAGCAACGATTCGAATGAGTTGTTCAATAATGATAGAGAAATGGGATGCATTGGTTTCAAGACGTGGAAACTCTGGGGAATTAGATGTTTGGCCTTATATTAGAGAACTATCAGGTGATATGATTTCTCGAGCAGCATTTGGAAGTAGTCATAAGGACGGAAGCAGAATATTTGAGCTCCAAAACGAGCAAGTGAAGCTCGTCTTGCAACTCTTGGCTTTCCATTTTATCCCTGGATGGAGTTATTTACCAACCAAGGCAAACAGAAAAATGAAA GCACTCGCCAAAGAAATTAAGTCCTTACTGAGGGGTATTATCGACCAGCGAGAGAAAGCAATGAAAAGGGGAGAAGAGATGGCATCTGATTTGTTGGGTATATTAATGGAGTCAAATTTCAAAGAAATGCAACAGCAAGGAAACAAGAAGATGGGAATGAGCATTGAGGATGTTGTAGAGGAATGCAAGCTGTTCTATATTGCAGGCTCTGATACCACTTCTAATTTGCTAGTGTGGACTATGGTAATGCTTAGTAAACATCCGGAATGGCAGGCTCGTGCAAGAGAAGAAGTTTTGCAAGTCTTTGGAAAGAGTGAACCAACTTTTGACGGTGTAAATCGCCTCAAAATCGTAACCATGATTCTTCAAGAAGTTCTCCGATTGTATC CACCAGTACCTTTGGTCCTCCGATCCCCTACAAAAGAGCTCAGACTGGGAGACATAACTTTGCCAAAGGACGTCGATGTAATTTTGTTAATGGGCATGCTCCATCATGATCCAGAAGTTTGGGGAGATGATGTACAAGACTTCAAACCTGAGAGATTTTCTGGAGGTATTTCATCCGCGGCAAAGACTCAATTCGCCTTCATGCCATTCGGCTTCGGACCTCGAATTTGCATAGGGCAAAATTTTGCAATGCTTGAGGCAAAAATCTCTTTGGCCATGATACTCCAGCGGTTTTCGTTTGAGTTGTCCTCGTCTTATTTGCATGCGCCTTTCCCCATTCTTACACTCGAACCACAGCATGGCGCTTCAATGGTTTTGCATAAACTATAG

SEQ ID NO.2

Amino Acid Sequence:

MAVKLSSILISLLVLALTTLVLKVLNWVWFRPKKLEKILRDQGLNGNPYRILIGDMKDLMSATNAEKSRPIQLSDDISQHMFPYYHQARSKYGPNSFVWFGPSPKLMIADPVLLKEIFTKPDVFHKPVPDPIGQSIAGGLTFLEDEKWAKHRRIINPAFHMEKLKDKTATIRMSCSIMIEKWDALVSRRGNSGELDVWPYIRELSGDMISRAAFGSSHKDGSRIFELQNEQVKLVLQLLAFHFIPGWSYLPTKANRKM KALAKEIKSLLRGIIDQREKAMKRGEEMASDLLGILMESNFKEMQQQGNKKMGMSIEDVVEECKLFYIAGSDTTSNLLVWTMVMLSKHPEWQARAREEVLQVFGKSEPTFDGVNRLKIVTMILQEVLRLYPPVPLVLRSPTKELRLGDITLPKDVDVILLMGMLHHDPEVWGDDVQDFKPERFSGGISSAAKTQFAFMPFGFGPRICIGQNFAMLEAKISLAMILQRFSFELSSSYLHAPFPILTLEPQHGASMVLHKL

SEQ ID NO.3

ATGGCAGTTAAATTGAGCAGC

SEQ ID NO.4

TTACAATTTGTGCAAAACCATAGAAGC

SEQ ID NO.5

cagtcgacctcgaatctaga ATGGCAGTTAAATTGAGCAGC

SEQ ID NO.6

ctaattacatgatgcggccc TTACAATTTGTGCAAAACCATAGAAGC

Claims (8)

1. A Bacopa monnieri P450 enzyme gene BmCYP068 , characterized in that the nucleotide sequence of the Bacopa monnieri P450 enzyme gene BmCYP068 is shown in SEQ ID NO.1. 2. The protein encoded by the Bacopa monnieri P450 enzyme gene BmCYP068 according to claim 1, characterized in that the amino acid sequence of the encoded protein is shown in SEQ ID NO.2. 3. A recombinant plasmid containing the Bacopa monnieri P450 enzyme gene BmCYP068 according to claim 1. 4. The recombinant plasmid containing the Bacopa monnieri P450 enzyme gene BmCYP068 according to claim 3, characterized in that the Bacopa monnieri P450 enzyme gene BmCYP068 is homologously recombined with a Y33 vector to obtain a Y33- BmCYP068 recombinant plasmid. 5. A genetically modified engineered bacterium, comprising the recombinant plasmid of claim 3, or the genome of the genetically modified bacterium is integrated with the exogenous Bacopa monnieri P450 enzyme gene BmCYP068 of claim 1. 6. The genetically modified engineered bacteria according to claim 5, characterized in that the genetically modified engineered bacteria are Dammarenediol II Saccharomyces cerevisiae chassis cells. 7. Use of the Bacopa monnieri P450 enzyme gene BmCYP068 according to claim 1 in the preparation of 23-OH-Dammarenediol II and 25-OH-Dammarenediol II. 8. Use of the Bacopa P450 enzyme gene BmCYP068 according to claim 7 in the preparation of 23-OH-Dammarenediol II and 25-OH-Dammarenediol II, characterized in that: Dammarenediol II produced by chassis cells is used as a substrate, and 23-OH-Dammarenediol II and 25-OH-Dammarenediol II are generated by hydroxylation at position 23 or 25 of the side chain under the catalysis of the Bacopa P450 enzyme BmCYP068 encoded by the Bacopa P450 enzyme gene BmCYP068 .