CN117625569A

CN117625569A - RrCYP450 protein, coding gene and application thereof

Info

Publication number: CN117625569A
Application number: CN202410108921.3A
Authority: CN
Inventors: 付玉杰; 宁愿; 丁运坤; 刘志国; 辛娣
Original assignee: Northeast Forestry University
Current assignee: Northeast Forestry University
Priority date: 2024-01-26
Filing date: 2024-01-26
Publication date: 2024-03-01
Anticipated expiration: 2044-01-26
Also published as: CN117625569B

Abstract

The invention provides RrCYP450 protein, a coding gene and application thereof, belonging to the technical field of synthetic biology. The invention uses the roxburgh rose to treat the diseasesRosa roxburghii Tratt.) For the study object, CYP450 enzyme protein catalyzing hydroxylation of C-2 site and C-19 site of pentacyclic triterpene compound is discovered for the first time by comprehensively utilizing transcriptomics and non-targeted metabonomics combination analysis: RCYP450-3, RCYP450-4 and RCYP450-5, and in vitro enzyme activity experiments prove that the RCYP450-3 and RCYP450-4 have the function of promoting the reaction at the ursolic acid C-2 sitePotential for hydroxylation, rrCYP450-5 has potential for hydroxylation at the ursolic acid C-19 site, and the hydroxylation modification function of three RrCYP450 in the Rosa roxburghii on pentacyclic triterpene is further verified through a molecular docking experiment.

Description

RrCYP450 protein, coding gene and application thereof

Technical Field

The invention belongs to the technical field of synthetic biology, and particularly relates to RrCYP450 protein, a coding gene and application thereof.

Background

CYP450s has large quantity, strong substrate specificity and low sequence similarity, and the physical properties of the expressed products are very similar and are difficult to separate, so that the difficulty of analyzing the bioconversion steps of the CYP450s is greatly increased. Classifying according to amino acid homology, CYP450 with amino acid homology >41% are assigned to the same family, CYP450 with amino acid homology >55% are considered to be the same subfamily, whereby CYP450 is divided into 11 clusters (Clan), 7 of which are each: CYP51, CYP74, CYP97, CYP10, CYP711, CYP727, and CYP746; there are 4 multi-family clusters, respectively: CYP71, CYP72, CYP85 and CYP86. Currently, 89 CYP450 genes with specific oxidation potential for pentacyclic triterpenes have been identified. By phylogenetic analysis of these CYP450s genes, it was found that CYP450s of different families, are clearly distinguished on the developmental tree, most of which have similar functions, e.g., CYP716A family members have C-28 oxidase activity, most of which catalyze the three consecutive steps of oxidation of the alpha-amyrin/beta-amyrin/lupeol backbone C-28 to form hydroxyl, aldehyde and carboxyl groups. Whereas members of the CYP716E and CYP716S families catalyze the hydroxylation of triterpenes C-6 and C-2 alpha, respectively, the CYP93E family catalyzes the oxidation of triterpenes mainly at the C-24 position. In addition, xu Yuanyuan et al summarized in plant triterpene saponin biosynthesis pathway and regulatory mechanism research progress that CYP450s also had the effect of catalyzing hydroxylation or carboxylation of the triterpene skeleton at positions C-3, C-11, C-12, C-16, C-19, C-20, C-21, C-22, C-23, C-25, C-30, etc. Through the oxidative modification of CYP450, the hydrophilic property of the triterpene compound is changed, and the activity and the functional efficacy of the triterpene component are increased.

The triterpene compounds have various structures, more than 100 different triterpene frameworks are generated by cyclizing the oxidosqualene, the first step of the structural diversity of the triterpene is caused, and the structural diversity of the triterpene compounds is finally caused by oxidation and glycosylation, so the characteristic is also the first difficulty in resolving the biosynthetic pathway of the triterpene compounds; in addition, in order to obtain the polyhydroxy triterpene compound with better activity, researchers are researching cytochrome P450 enzyme (CYP 450 enzyme), the CYP450 enzyme has huge family, strong substrate specificity and low sequence similarity, and the expressed products have very similar physical properties and are difficult to separate, so that the difficulty of analyzing the bioconversion step of the polyhydroxy triterpene compound is greatly increased, and the second difficulty of analyzing the biosynthesis path of the triterpene compound is caused.

Therefore, the problem can be greatly solved by excavating the gene with the specific modification function for the biosynthesis of the triterpene compound, and the analysis of the key synthesis steps of the polyhydroxy triterpene compound is further realized.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides RCYP450 protein, a coding gene and application thereof, wherein the RCYP450 protein is from roxburgh rose, is an enzyme protein for hydroxylation of C-2 or C-19 of pentacyclic triterpene compounds, can provide sites for further glycosylation and acylation to form triterpene compounds with various structures, provides important references for researching hydroxylation modification of the pentacyclic triterpene compounds, can carry out hydroxylation of C-2 and C-19 sites in vitro by taking ursolic acid as a substrate, expands discovery and identification of plant CYP450 genes, and plays a vital role in exploration of triterpene biosynthesis.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

the first object of the present invention is to provide an RCYP450 protein combination for synthesizing roxburgh rose acid, wherein the RCYP450 protein combination consists of RCYP450-3 and RCYP450-5, or consists of RCYP450-4 and RCYP450-5, the amino acid sequence of the RCYP450-3 is shown as SEQ ID NO.1, the amino acid sequence of the RCYP450-4 is shown as SEQ ID NO.2, and the amino acid sequence of the RCYP450-5 is shown as SEQ ID NO. 3.

A second object of the present invention is to provide the use of a gene combination consisting of an RrCYP450-3 gene and an RrCYP450-5 gene, or an RrCYP450-4 gene and an RrCYP450-5 gene, for expressing the above protein combination, wherein the nucleotide sequence of the RrCYP450-3 gene is as set forth in SEQ ID NO:4, the base sequence of the RrCYP450-4 gene is shown in SEQ ID NO:5, the base sequence of the RrCYP450-5 gene is shown as SEQ ID NO: shown at 5.

The third object of the present invention is to provide the use of the above RCYP450-3 or the above RCYP450-3 gene for catalyzing hydroxylation of C-2 position in pentacyclic triterpene compounds.

The fourth object of the present invention is to provide the use of the above RCYP450-4 or the above RCYP450-4 gene for catalyzing hydroxylation of C-2 position in pentacyclic triterpene compounds.

A fifth object of the present invention is to provide the use of the RrCYP450-5 described above or the RrCYP450-5 gene described above for catalyzing hydroxylation of the C-19 position in pentacyclic triterpene compounds.

It is a sixth object of the present invention to provide a recombinant expression vector, a recombinant expression cell or a recombinant expression strain comprising the above-mentioned RrCYP450-3 gene.

The seventh object of the present invention is to provide a recombinant expression vector, a recombinant expression cell or a recombinant expression strain comprising the RrCYP450-4 gene described above.

An eighth object of the present invention is to provide a recombinant expression vector, a recombinant expression cell or a recombinant expression strain comprising the above-mentioned RrCYP450-5 gene.

Further, the pentacyclic triterpene compound is ursolic acid.

The ninth object of the present invention is to provide the preparation method of the protein RrCYP450-3 or RCYP450-4 or RCYP450-5, connecting linearized pET28 (a+) -SUMO plasmid with RCYP450-3 or RCYP450-4 or RCYP450-5 gene respectively, constructing corresponding recombinant expression vectors, respectively introducing the recombinant expression vectors into an escherichia coli host cell to obtain corresponding recombinant expression cells, culturing the recombinant expression cells, and obtaining the protein RrCYP450-3 or RCYP450-4 or RCYP450-5 from the culture.

The term "expression" as used in the present invention includes any step involving the production of a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

The term "recombinant vector" as used in the present invention means a linear or circular DNA molecule comprising a polynucleotide encoding a polypeptide operably linked to control sequences for its expression.

The term "host cell" as used in the present invention means any cell type that is readily transformed, transfected, transduced, or the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

Compared with the prior art, the technical scheme provided by the invention has the beneficial effects that:

the invention uses the roxburgh rose to treat the diseasesRosa roxburghii Tratt.) For the study object, CYP450 enzyme protein catalyzing hydroxylation of C-2 site and C-19 site of pentacyclic triterpene compound is discovered for the first time by comprehensively utilizing transcriptomics and non-targeted metabonomics combination analysis: RCYP450-3, RCYP450-4 and RCYP450-5, and in vitro enzyme activity experiments prove that RCYP450-3 and RCYP450-4 have the potential of hydroxylation at the ursolic acid C-2 site, RCYP450-5 has the potential of hydroxylation at the ursolic acid C-19 site, and after RCYP 450-4+RCYP 450-5 and RCYP 450-3+RCYP 450-5 are combined and reacted, the specific pentacyclic triterpenoid rosary pear acid of the rosa roxburghii can be produced respectively, and the specificity is good; the hydroxylation modification function of three RrCYP450 in the Rosa roxburghii on pentacyclic triterpene is further verified through a molecular docking experiment.

Drawings

FIG. 1 is a schematic diagram of the plasmid structure of pET28a-SUMO-RrCYP450-3 expression vector;

FIG. 2 is a schematic diagram of the plasmid structure of pET28a-SUMO-RrCYP 450-4 expression vector;

FIG. 3 is a schematic diagram of the plasmid structure of pET28a-SUMO-RrCYP 450-5 expression vector;

FIG. 4 is a polyacrylamide gel electrophoresis of RrCYP450, wherein '1' represents a wild type control group, '2' represents an empty control group, and '3' represents a protein expression group;

FIG. 5 is a graph showing the results of physicochemical properties of RrCYP 450;

fig. 6 and 7 are graphs of the results of in vitro enzyme activity detection of RrCYP450, fig. a: ursolic acid control, B: rrCYP450-4, C: rrCYP450-3, D: rrCYP450-5, E: rosc acid control, F: rrCYP450-4+RrCYP450-5, G: rrCYP450-3+RrCYP450-5;

FIG. 8 is a molecular docking diagram of RrCYP450-3 and a substrate ursolic acid;

FIG. 9 is a molecular docking diagram of RrCYP450-4 and a substrate ursolic acid;

FIG. 10 is a molecular docking diagram of RrCYP450-5 and a substrate ursolic acid.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the following detailed description of the specific embodiments of the present invention will be given with reference to the accompanying drawings. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The amino acid sequence of the RrCYP450 protein provided by the invention is any sequence shown in SEQ ID NO. 1-3. Wherein the protein shown in SEQ ID NO.1 is denoted as RCYP450-3, the protein shown in SEQ ID NO.2 is denoted as RCYP450-4, and the protein shown in SEQ ID NO.3 is denoted as RCYP450-5.

The nucleotide sequence of the encoding gene of the protein shown in SEQ ID NO.1 is shown as SEQ ID NO. 4; the nucleotide sequence of the coding gene of the protein shown in SEQ ID NO.2 is shown in SEQ ID NO. 5; the nucleotide sequence of the coding gene of the protein shown in SEQ ID NO.3 is shown in SEQ ID NO. 6. In one embodiment, the polynucleotide encoding a protein of the invention has been isolated. Techniques for isolating or cloning polynucleotides are known in the art and include isolation from genomic DNA or cDNA or a combination thereof. Cloning of polynucleotides from genomic DNA can be accomplished, for example, by using the well-known Polymerase Chain Reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments having shared structural features. See, e.g., innis et al, 1990, PCR: AGuidetomethodsandApplication [ PCR: methods and application guide ], academic Press (academic Press), new York. Other nucleic acid amplification procedures such as Ligase Chain Reaction (LCR), ligation Activated Transcription (LAT) and polynucleotide-based amplification (NASBA) may be used.

The invention also relates to recombinant vectors for constructing the above-described coding genes, which comprise a polynucleotide of the invention operably linked to one or more control sequences that direct the expression of the coding sequences in a suitable host cell under conditions compatible with the control sequences. The polynucleotides can be manipulated in a number of ways to provide for expression of polypeptides. Depending on the expression vector, it may be desirable or necessary to manipulate the polynucleotide prior to insertion into the vector. Techniques for modifying polynucleotides using recombinant DNA methods are known in the art. The control sequence may be a promoter, i.e., a polynucleotide recognized by a host cell for expression of a polynucleotide encoding a polypeptide of the invention. The promoter contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter may be any polynucleotide that exhibits transcriptional activity in the host cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

In some embodiments, it also relates to recombinant expression vectors comprising a polynucleotide of the invention, a promoter, and transcriptional and translational stop signals. Multiple nucleotides and control sequences may be linked together to produce a recombinant expression vector, which may include one or more convenient restriction sites to allow for insertion or substitution of the polynucleotide encoding the polypeptide at these sites. Alternatively, the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression. In generating the expression vector, the coding sequence is located in the vector such that the coding sequence is operably linked to the appropriate control sequences for expression. The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and that can cause expression of the polynucleotide. The choice of vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.

In some embodiments, it also relates to recombinant host cells comprising a polynucleotide of the invention operably linked to one or more control sequences that direct the production of a polypeptide of the invention. The construct or vector comprising the polynucleotide is introduced into a host cell such that the construct or vector is maintained as a chromosomal integrant or as an autonomously replicating extra-chromosomal vector, as described earlier. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of host cell will depend to a large extent on the gene encoding the polypeptide and its source. The host cell may be any cell useful in the recombinant production of the polypeptides of the invention, such as a prokaryotic cell or a eukaryotic cell.

Example 1

This example provides the preparation of RrCYP450 proteins.

1 Synthesis of Gene

1.1 Extraction of RNA and synthesis of cDNA.

Rosa roxburghii leaf source: the inventor acquires the medicine in 2019 in Miao nationality of Qian, guizhou, brillouin county.

Extracting total RNA of Rosa roxburghii leaf, synthesizing cDNA immediately with qualified total RNA of Rosa roxburghii leaf, and storing the rest in a refrigerator at-80deg.C for use.

cDNA synthesis was amplified using PrimeScript ™ II 1st Strand cDNA Synthesis Kit kit for subsequent amplification of the full field sequence of the target gene. Preparing a reaction system according to the steps of a reagent kit product specification, fully and uniformly mixing the prepared system, centrifuging, putting the system into a PCR amplification instrument for reaction, and setting a reaction program as follows:

(1) The reaction was carried out at 65℃for 5 min in accordance with the reaction system shown in Table 1-1 below, followed by rapid cooling on ice;

table 1-1.

(2) According to the reaction system shown in the following tables 1-2, 42 ℃ (50 ℃) for 30-60 min; after 5 min at 95 ℃ (enzyme inactivation), cooling on ice (4 ℃);

tables 1-2.

1.2 Small amount PCR amplification system and reaction condition

(a) Amplification was performed using PrimeSTAR GXL DNA polymerase and the PCR reaction system is shown in tables 1-3 (40 μl/tube):

tables 1-3.

The PCR reaction conditions (30 reaction cycles) are shown in the following tables 1 to 4:

tables 1 to 4.

(b) The PCR reaction system (10. Mu.L/tube) was amplified using Taq DNA polymerase as shown in tables 1-5 below:

tables 1 to 5.

The PCR reaction conditions (30 reaction cycles) are shown in the following tables 1 to 6:

tables 1 to 6.

(c) The drop PCR reaction conditions (30 reaction cycles) are shown in tables 1-7 below:

tables 1 to 7.

1.3 gel recovery of DNA fragments

The Omega agarose gel DNA recovery kit is adopted, and specific operation is shown in the specification of the Omega agarose gel DNA recovery kit.

2. Construction of recombinant expression plasmids

In the present invention, cloning methods for constructing plasmid vectors include Gibson assembly and. Gison assembly, the study was performed using the Hieff CloneTM Plus Multi One Step Cloning Kit kit from Saint Corp, which was operated according to the kit instructions.

2.1 Cleavage of vector plasmid Using the cleavage System the following Table 2-1 shows:

table 2-1.

2.2 Linearization of vector plasmid to obtain donor DNA

The donor DNA was obtained by cleavage with NotI, the cleavage system being shown in Table 2-2:

table 2-2.

2.3 Small-scale extraction of E.coli plasmid DNA

Plasmid DNA miniprep kit of Axygen company is selected as plasmid extraction. The specific operation process is shown in the instruction of the Axygen plasmid DNA small-scale extraction kit, and the small-scale extraction of the escherichia coli plasmid DNA can be completed.

2.4 Construction of expression vectors

The Gibson assembly is carried out by selecting a Hieff CloneTM Plus Multi One Step Cloning Kit kit of assist Saint company, and the specific method is as follows:

firstly, obtaining a CDS sequence of a target gene through PCR amplification, wherein the 5' end of a primer contains an overlapping sequence of 60-80 bp when designing the primer of the target gene; after electrophoresis determines the target strip, cutting glue to recycle PCR product and measuring concentration; preparing a reaction system according to the requirements of a kit instruction, uniformly mixing and centrifuging, and then placing the mixture in a PCR amplification instrument for incubation at 50 ℃ for 50 min; after the reaction is finished, the method can be used for converting escherichia coli competent cells BL21 (DE 3) after ice bath cooling.

According to the RrCYP450 gene obtained by screening, a primer design is carried out by using PrimePrime5, and the Rosa roxburghii cDNA is used as a template, and 4 full-length sequences of the RrCYP450 gene are obtained by PCR amplification, namely RrCYP450-1, rrCYP450-3, rrCYP450-4 and RrCYP450-5, and are synthesized by Shanghai bioengineering Co Ltd. The expression vector of corresponding RrCYP450 escherichia coli is constructed by connecting linearized pET28 (a+) -SUMO plasmid with RrCYP450 genes by using assist in the sea, wherein the total of 3 RrCYP450 genes are used for successfully constructing the expression vector, and the construction result of the vector is shown in figures 1-3.

Expression of the 3 RrCYP450 protein

3.1 Competent preparation of E.coli

E.coli BL21 (DE 3) strain is taken to be streaked on a LA plate, and after single colony grows out, about 10 single colonies are picked out and transferred into 5 mL LB liquid medium for overnight culture. Transferring 2 mL strain solution into 200 mL newly prepared LB liquid medium, shake culturing to OD ₆₀₀ The value is 0.3-0.5. The bacterial liquid was removed, transferred to a sterilized 50 mL centrifuge tube, centrifuged at 3000rpm and 4℃for 10 min, and the supernatant was discarded. 80 mL of pre-chilled T1 solution at 4℃was added, gently sucked, resuspended and ice-bathed for 30 min.3000 After centrifugation at rpm at 4℃for 10 min, the supernatant was discarded. Solution T1 was repeatedly washed three times. 8 mL of pre-cooled T2 solution at 4 ℃ is added, and the mixture is fully resuspended, thus obtaining DH10B competent cells. The DH10B competent cells were packed in sterile centrifuge tubes on ice and snap frozen immediately and stored at-80 ℃.

3.2 Competent transformation of E.coli

Competent E.coli BL21 (DE 3) cells were taken and thawed on ice. Plasmid 10 ng was added to the freshly thawed competent cells and gently mixed. After ice bath for 30 min, heat shock 90 s is carried out at 42 ℃, and then ice bath is carried out rapidly for 3 min. About 800. Mu.L of LB medium was added to the above system and mixed well. Shaking culture for 45-60 min.4000 Centrifugation at rpm for 2-3 min, discarding supernatant, resuspending about 200. Mu.L of supernatant, spreading on LA plates of corresponding resistance, and culturing overnight in inversion.

Plasmid was prepared by: pET28a (+) -SUMO-RrCYP450-5, pET28a (+) -SUMO-RCYP 450-3, pET28a (+) -SUMO-RCYP 450-4 were transformed into E.coli BL21 (DE 3) competence for protein expression. And (3) after IPTG low-temperature induction, collecting thalli at a low temperature. The collected cells were disrupted by an ultrasonic disrupter to disrupt E.coli cells, and then centrifuged at 12000 rpm for 40 min to collect the supernatant, which was a supernatant containing the target protein, i.e., crude protein. The crude protein solution was subjected to protein concentration detection and SDS-PAGE electrophoresis analysis.

3.2.1 Protein concentration determination by BCA method

The detection of the crude enzyme protein concentration is carried out by using a BCA protein concentration kit produced by Shanghai Biyun biotechnology Co-Ltd, and specific operation steps are shown in a specification in the kit. Standard curves were drawn using standard protein solutions: y=0.0566 x+ 0.2869 (R ² =0.9993), the concentration of the crude enzyme was calculated by substituting the measured absorbance value of the crude enzyme into the equation.

3.2.2 SDS-PAGE electrophoretic analysis

And (3) regulating the concentration of the sample according to the quantitative result of the protein concentration, adding a loading buffer solution, boiling for 5 min, and centrifuging at 3000rpm for 5 min to obtain the loading protein. The gel was prepared using SDS-PAGE gel preparation kit. The position and expression of the target band can be determined by SDS-PAGE analysis. The main procedure of SDS-PAGE electrophoresis is as follows:

and (3) glue preparation: and assembling the glue making device, respectively adding separating glue and concentrated glue into gaps of two glass plates by using a liquid transfer device according to the requirements of the kit specification, inserting a comb, and loading the glue after the upper glue layer and the lower glue layer form an obvious interface (about 20 minutes), wherein the glue can be completely polymerized.

Loading: and (3) assembling the protein electrophoresis device, adding the prepared 1 XTris-glycine electrophoresis buffer solution into the electrophoresis tank, pulling out the comb, and adding the sample according to the protein loading amount of 20 mu L/each hole by using a pipette.

Electrophoresis: setting the voltage of the sample to 80V when the sample is electrophoresed in the concentrated gel, changing the sample into 100V when the sample enters the separation gel, and ending electrophoresis until bromophenol blue reaches the bottom of the gel.

Dyeing: the separation gel was stained with coomassie brilliant blue fast stain for 30 min, and after staining, destaining and observation were performed with distilled water.

The BCA protein concentration detection kit is adopted to quantitatively obtain three groups of crude protein concentrations of RrCYP450-3, rrCYP450-4 and RrCYP450-5 of crude enzyme, wherein the three groups of crude protein concentrations are respectively as follows: 6.09 mg/L, 4.307 mg/L, 3.909 mg/L. And carrying out SDS-PAGE polyacrylamide gel electrophoresis on the three groups of crude proteins and the control thereof to obtain a result shown in figure 4, wherein the protein electrophoresis obviously shows that the three groups of proteins are obviously expressed after IPTG induction, and the molecular weight of the target protein is about 42 kD, so that the target protein is confirmed.

Example 2

This example provides a study of the in vitro enzymatic activity of the RrCYP450 protein.

1. Reaction system condition determination for in vitro enzyme activity experiment

The protein RrCYP450-3, rrCYP450-4 and RrCYP450-5 are subjected to protein physicochemical property analysis, and analysis results are shown in a figure 5, which shows that 3 CYP450 proteins are all alkalescent stable proteins and all have a transmembrane structure and a signal peptide. Therefore, when preparing a reaction system, a slightly alkaline environment is selected for reaction.

The reaction system was determined to be configured as shown in table 3:

table 3.

The reaction was carried out at 28℃and was stopped by adding 70% methanol at a final concentration after the completion of the reaction, reaction 12 and h.

2. Analysis of results of in vitro enzyme Activity experiments

HPLC detection method: samples were analyzed using a sammer fly Vanquish liquid chromatograph. The mobile phase is water (a): chromatographic acetonitrile (C), a chromatographic column of HiQ sil C18W (4.6 mm phi multiplied by 250 mm) is selected, and the sample injection amount is 10 mu L; the flow rate is 1mL/min; the column temperature is 30 ℃; the detection wavelength is λ=210 nm. Chromeleon 7 workstation software was used for data acquisition and analysis.

The reaction system was subjected to HPLC detection, and the detection results are shown in FIGS. 6 and 7.

In an in vitro enzyme activity experiment, three RrCYP450 enzymes all catalyze ursolic acid and produce corresponding catalytic products, wherein RrCYP450-4 and RrCYP450-3 with C-2 modification potential detect the product compound 1 within the retention time t=18.25 min, and RrCYP450-5 with C-19 modification potential detects the product compound 2 within the retention time t=17.5 min. After RrCYP450 with C-2 and C-19 modification potential is respectively combined, HPLC detection is carried out, and after comparison with a roxburgh rose acid reference substance, rrCYP450-4+RrCYP450-5 and RrCYP450-3+RrCYP450-5 reaction systems are identified to generate roxburgh rose acid, and meanwhile, a byproduct compound 3 is generated within the retention time t=11.52 min.

The detection result shows that RCYP450-3 gene, RCYP450-4 gene and RCYP450-5 gene in Rosa roxburghii have the capacity of carrying out hydroxylation modification on ursolic acid C-2 and C-19.

When RrCYP450-4+RrCYP450-5 and RrCYP450-3+RrCYP450-5 are combined and reacted, the specific pentacyclic triterpene rosa roxburghii acid can be generated. In addition, the invention also uses oleanolic acid as a substrate to respectively carry out in vitro activity experiments with three RCYP450 enzymes, and experimental results show that the three RCYP450 enzymes have almost no catalytic activity in vitro when being aligned with the oleanolic acid.

Example 3

Homology modeling of RrCYP450 proteins is in molecular docking.

RCYP450-3, RCYP450-4 and RCYP450-5 homology models are constructed by adopting a SWISS model server (http:// swissmodel. Expasy. Org /). Searching and comparing in a protein library, and selecting three structural reference models of proteins as follows: a0a540l0s2.1.A, a0a2p6q8z0.1.A, a0a540kzj1.1.A.

Discovery Studio 2016 Client is used for model optimization and model structure interfacing. To ensure that all models were of good quality, the model z-score was calculated and the calculation found that >90% of the residues were within the range of the native folding protein, with a reliable model. A grid box comprising 18 x 18 or 20 x 20 is arranged when in butt joint, wherein the active pocket contains RrCYP450-3, rrCYP450-4 and RrCYP450-5, as a search space, molecular model docking analysis is carried out with a docking substrate ursolic acid. Molecular docking results were processed using liglot visualization software as shown in fig. 8-10: FIG. 8 shows a molecular docking model between RrCYP450-3 and ursolic acid, wherein Arg343 and Ala107 amino acid residues are arranged at the C-2 position of the ursolic acid structure to generate binding activity, FIG. 9 shows a molecular docking model between RrCYP450-4 and ursolic acid, gly210 and Leu211 amino acid residues are arranged at the C-2 position of the ursolic acid structure to generate binding activity, and FIG. 10 shows a molecular docking model between RrCYP450-5 and ursolic acid, and His456 amino acid residues are arranged at the C-19 position of the ursolic acid structure to generate binding activity. The result of molecular docking further proves the hydroxylation modification function of three RrCYP450 in the Rosa roxburghii on pentacyclic triterpene.

The embodiments described above and features of the embodiments herein may be combined with each other without conflict.

The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.

Claims

1. The RCYP450 protein combination for synthesizing the roxburgh rose acid is characterized by comprising RCYP450-3 and RCYP450-5 or comprising RCYP450-4 and RCYP450-5, wherein the amino acid sequence of the RCYP450-3 is shown as SEQ ID NO.1, the amino acid sequence of the RCYP450-4 is shown as SEQ ID NO.2, and the amino acid sequence of the RCYP450-5 is shown as SEQ ID NO. 3.

2. Use of a gene combination for expressing the RrCYP450 protein combination according to claim 1 in the synthesis or production of roxburgh rose acid, wherein the gene combination consists of a RrCYP450-3 gene and a RrCYP450-5 gene, or consists of a RrCYP450-4 gene and a RrCYP450-5 gene, the base sequence of the RrCYP450-3 gene is shown as SEQ ID No.4, the base sequence of the RrCYP450-4 gene is shown as SEQ ID No.5, and the base sequence of the RrCYP450-5 gene is shown as SEQ ID No. 6.

3. Use of RrCYP450-3 as defined in claim 1 or of a RrCYP450-3 gene as defined in claim 2 for catalyzing hydroxylation at position C-2 in pentacyclic triterpene compounds.

4. Use of RrCYP450-4 as defined in claim 1 or of the RrCYP450-4 gene as defined in claim 2 for catalyzing hydroxylation at position C-2 in pentacyclic triterpene compounds.

5. Use of RrCYP450-5 as defined in claim 1 or of a RrCYP450-5 gene as defined in claim 2 for catalyzing hydroxylation at position C-19 in pentacyclic triterpene compounds.

6. A recombinant expression vector, recombinant expression cell or recombinant expression strain comprising the RrCYP450-3 gene of claim 2.

7. A recombinant expression vector, recombinant expression cell or recombinant expression strain comprising the RrCYP450-4 gene of claim 2.

8. A recombinant expression vector, recombinant expression cell or recombinant expression strain comprising the RrCYP450-5 gene of claim 2.

9. The use according to any one of claims 3 to 5, wherein the pentacyclic triterpene compound comprises ursolic acid.

10. The method for preparing the protein RrCYP450-3 or RrCYP450-4 or RrCYP450-5 according to claim 1, wherein the linearized pET28 (a+) -SUMO plasmid is respectively connected with the RrCYP450-3 or RrCYP450-4 or RrCYP450-5 gene to construct corresponding recombinant expression vectors, the recombinant expression vectors are respectively introduced into escherichia coli host cells to obtain corresponding recombinant expression cells, the recombinant expression cells are cultured, and the protein RrCYP450-3 or RrCYP450-4 or RrCYP450-5 is obtained from the culture.