Phryma leptostachya CYP706AA oxygenase gene and application thereof
By digging out the CYP706AA oxygenase gene in the periarthritis and building a recombinant expression system, the shortcomings in the production and application of periarthritis compounds in the prior art were solved, and efficient production of periarthritis V with antiviral activity was achieved.
Patent Information
- Application Number
- CN202510291944.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-12
- Publication Date
- 2025-07-22
AI Technical Summary
In the prior art, there is a lack of in-depth research on the specific mechanism of the oxygenase gene in the permeabilata and its encoded protein function, resulting in a lack of new ways to produce and apply the permeabilata compounds.
The CYP706AA oxygenase gene in the pericardium was excavated by genomics, metabolomics and transcriptomics, and a recombinant expression plasmid was constructed and the pericarium oxygenase protein was expressed in Saccharomyces cerevisiae. It was used as a biocatalyst to catalyze the pericarium oxygenase gene to generate pericarium virgin V.
The specific sequence of the oxygenase gene and its role in catalyzing the oxygen-entrapped pauillon to produce pervious ceramide V were clarified, and the efficient production of pervious ceramide compounds with antiviral activity was achieved.
Smart Images
Figure CN120350035A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of plant genetic engineering and relates to the CYP706AA oxygenase gene of Speranskia tuberculata and its application. Background Art
[0002] As a traditional medicinal plant, Speranskia tuberculata is widely used for insecticidal and medicinal purposes in Southeast Asia. Structurally diverse neolignan compounds isolated from Speranskia tuberculata have been proven to have insecticidal synergistic effects. The formation of the oxygen insertion structure is of great significance for the structural diversity and biological activity of its lignan compounds. The identified phyllanthurinolactone I, phyllanthurinolactone II, phyllanthurinolactone III, phyllanthurinolactone IV, and phyllanthurinolactone V isolated from Speranskia tuberculata all contain this oxygen insertion structure. At the same time, studies have shown that it may be this oxygen insertion structure that endows the phyllanthurinolactone compounds with antiviral activity.
[0003] However, there is currently a lack of in-depth research on the specific mechanism of action of the oxygenase gene in Speranskia tuberculata and the function of the protein encoded by it.
[0004] Therefore, in the technical field of plant genetic engineering, through in-depth research on the specific mechanism of action of the oxygenase gene in Speranskia tuberculata and the function of the protein encoded by it, new ways can be provided for the production and application of phyllanthurinolactone compounds. Summary of the Invention
[0005] The present invention combines genomics, metabolomics, and transcriptomics to mine and screen the CYP706AA oxygen insertion gene in Speranskia tuberculata, and obtains the Speranskia tuberculata oxygenase gene and the Speranskia tuberculata oxygenase protein. The Speranskia tuberculata oxygenase protein can catalyze the oxygen insertion between the carbon-carbon bonds of the benzene ring and the furan ring of paulownin.
[0006] To achieve this technical purpose, the present invention adopts the following technical solutions.
[0007] In a first aspect, the present invention provides a Speranskia tuberculata CYP706AA oxygenase gene, and the Speranskia tuberculata CYP706AA oxygenase gene includes the CYP706AA7 gene and the CYP706AA14 gene, wherein,
[0008] The CYP706AA7 gene:
[0009] a1) Encodes the CYP706AA7 protein;
[0010] a2) Has the nucleotide sequence shown in SEQ ID NO: 1;
[0011] The CYP706AA14 gene:
[0012] b1) Encodes the CYP706AA14 protein;
[0013] b2) It has the nucleotide sequence shown in SEQ ID NO:2.
[0014] In a second aspect, the present invention provides a Phryma leptostachya CYP706AA oxygenase protein, which includes CYP706AA7 protein and CYP706AA14 protein. Among them,
[0015] The CYP706AA7 protein:
[0016] A1) It has the amino acid sequence shown in SEQ ID NO:3;
[0017] A2) A derived amino acid sequence obtained by substituting, deleting and / or adding one or several amino acids to the amino acid sequence in A1), and the derived amino acid sequence has the activity of the amino acid sequence shown in A1); or
[0018] A3) An amino acid sequence having at least 80% homology with the amino acid sequence in A1);
[0019] The CYP706AA14 protein:
[0020] B1) It has the amino acid sequence shown in SEQ ID NO:4;
[0021] B2) A derived amino acid sequence obtained by substituting, deleting and / or adding one or several amino acids to the amino acid sequence in B1), and the derived amino acid sequence has the activity of the amino acid sequence shown in B1); or
[0022] B3) An amino acid sequence having at least 80% homology with the amino acid sequence in B1).
[0023] In a third aspect, a recombinant expression plasmid containing the Phryma leptostachya CYP706AA oxygenase gene as described in the present invention is provided.
[0024] Preferably, the original plasmid used for the recombinant expression plasmid is pYES2 plasmid.
[0025] Preferably, the recombinant expression plasmids are pYES2-PlCYP706AA7 and
[0026] pYES2-PlCYP706AA14.
[0027] Preferably, the CYP706AA7 gene and the CYP706AA14 gene are inserted between the BamH I and Hind III sites of the original plasmid.
[0028] Fourthly, a recombinant strain expressing the CYP706AA oxygenase gene of Speranskia tuberculata as described in the present invention is provided. The recombinant strain uses Saccharomyces cerevisiae BY4742 as the host bacterium and transforms the recombinant expression plasmid as described in the present invention.
[0029] Fifthly, the application of the CYP706AA oxygenase protein of Speranskia tuberculata as described in the present invention as a biocatalyst is provided.
[0030] Preferably, the application process of the CYP706AA oxygenase protein of Speranskia tuberculata is as follows: The reaction is carried out in a reaction system containing a reaction substrate, NADPH, yeast microsomal protein encoded by the CYP706AA oxygenase gene of Speranskia tuberculata, and Tris-HCl buffer.
[0031] Preferably, the reaction time is 2 h, the reaction temperature is 30 °C, the reaction pH is 7.2, and the reaction system is 300 μL.
[0032] Preferably, the reaction substrate is paulownin.
[0033] In the present invention, the reaction substrate is prepared into a standard stock solution of 100 mM with DMSO before use, the final concentration of the reaction substrate used is 100 μM, and the concentration of NADPH used is 500 μM; the dosage of the yeast microsomal protein encoded by the CYP706AA oxygenase gene of Speranskia tuberculata is 50 μL, the concentration of the Tris-HCl buffer is 100 mM, and the pH is 7.2.
[0034] In the present invention, the termination reaction reagent after the reaction is ethyl acetate, and the dosage of the ethyl acetate is 600 μL.
[0035] In the present invention, after the termination of the reaction, liquid-liquid extraction and centrifugal stratification are carried out. The rotation speed of the centrifugation is 12000 rpm; the centrifugation time is 3 min.
[0036] In the present invention, the upper ethyl acetate phase is transferred to a new 1.5 mL centrifuge tube and placed in a vacuum concentrator until the ethyl acetate is completely volatilized. The residue in the centrifuge tube is resuspended with 55% acetonitrile water and fixed to 200 μL. The obtained sample solution is filtered through a 0.22 μm filter, and a clear and transparent reaction system resuspension is obtained after filtration, waiting for liquid phase detection.
[0037] Sixthly, the application of the CYP706AA oxygenase protein of Speranskia tuberculata in the oxygenation of paulownin to generate speranskialactone V is provided.
[0038] In the present invention, the term "nucleotide sequence" or "polynucleotide" refers to a linear polymer of natural or synthetic nucleotide residues linked by phosphodiester bonds or their analogs. It can be single-stranded or double-stranded and includes RNA, DNA (e.g., it can be genomic, cDNA, or synthetic), its analogs, or combinations thereof. Those skilled in the art should understand that the representation order of nucleotide sequences is generally in the 5'-3' order from left to right, and, unless otherwise indicated, "A" refers to deoxyadenosine, "C" refers to deoxycytidine, "G" refers to deoxyguanosine, "T" refers to deoxythymidine, and "U" refers to ribonucleoside, uridine. Generally, nucleotide sequences contain four natural deoxynucleotides or four natural ribonucleotides; however, they can also contain unnatural nucleotide analogs.
[0039] In the present invention, the term "amino acid sequence" refers to the arrangement order of amino acids in a protein. This sequence is determined by genetic information, specifically, it is determined by genes in DNA or RNA through the processes of transcription and translation. Amino acid sequences are usually represented by strings of letters, with each letter representing a specific amino acid. These sequences are arranged in the direction from the N-terminus (amino terminus) to the C-terminus (carboxyl terminus). In biology, there are 20 different standard amino acids, and each amino acid has a specific single-letter or three-letter abbreviation. For example, the three-letter and single-letter abbreviations of lysine are Lys and K, respectively.
[0040] In the present invention, the term "gene" is the main substance of genetic variation. It is the basic unit controlling traits and is composed of the arrangement order of many different base pairs on deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules that determine biological traits. Genes express the genetic information they carry by guiding the synthesis of proteins, thereby controlling the trait manifestations of biological individuals. Specifically, genes are located on chromosomes, arranged linearly, and are genetic units occupying a certain position on chromosomes. Genes have duality, that is, they can faithfully replicate themselves to maintain the basic characteristics of organisms; at the same time, they may mutate during the reproduction of offspring. Mutated genes can also continue to replicate and be passed on to offspring. Genes can not only transmit genetic information to the next generation through replication but also express the genetic information in the molecular structure of proteins. Therefore, genes are the basic units controlling traits. A gene consists of only one DNA molecule, but one DNA molecule contains multiple genes. Genes are segments on DNA molecules with genetic effects, and they determine the expression of traits.
[0041] In the present invention, the term "protein" refers to a protein, which is the material basis of life, an organic macromolecule, the basic organic matter that constitutes cells, and the main bearer of life activities. Without proteins, there is no life. Amino acids are the basic building blocks of proteins. It is a substance that is closely linked to life and various forms of life activities.
[0042] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0043] (1) Mining and screening the epoxygenase genes in Phryma leptostachya L.: By comprehensive methods of genomics, metabolomics and transcriptomics, key epoxygenase genes are mined and screened from Phryma leptostachya L., especially members of the CYP706AA family, to obtain the CYP706AA7 gene and the CYP706AA14 gene.
[0044] (2) Analyzing the functions of the epoxygenase genes and their encoded proteins: clarifying the specific sequences of these epoxygenase genes and the roles of the proteins they encode in the process of catalyzing the epoxidation of paulownin to generate phrymarolin compounds. Specifically, the epoxygenase protein of Phryma leptostachya L. in the present invention is used for the epoxidation of paulownin to generate phrymarolin V.
[0045] (3) Constructing an efficient expression system: constructing recombinant plasmids containing these epoxygenase genes and expressing them in suitable host cells (Saccharomyces cerevisiae BY4742 host cells) to achieve the efficient production of epoxygenase proteins.
[0046] (4) Developing the applications of epoxygenase proteins: using these epoxygenase proteins as biocatalysts to catalyze the epoxidation of paulownin to generate phrymarolin compounds with antiviral activity. Specifically, the epoxygenase protein of Phryma leptostachya L. in the present invention is used for the epoxidation conversion of paulownin to phrymarolin V.
[0047] In summary, the present invention aims to solve the problems existing in the prior art by mining and screening the epoxygenase genes in Phryma leptostachya L., analyzing the functions of their encoded proteins, constructing an efficient expression system, and developing the applications of epoxygenase proteins, so as to provide a new way for the production and application of phrymarolin compounds. BRIEF DESCRIPTION OF THE DRAWINGS
[0048] Figure 1 It is a localization map of PlCYP706AA7 (CYP706AA7 gene) and PlCYP706AA14 (CYP706AA14 gene) on the chromosome.
[0049] Figure 2 It is the predicted three-dimensional structure of the proteins of PlCYP706AA7 and PlCYP706AA14.
[0050] Figure 3Sequence alignment of PlCYP706AA7 and PlCYP706AA14 with SmCYP76AH1 and SmCYP76AH3 whose functions have been characterized in Salvia miltiorrhiza, and IrCYP706V2 and IrCYP706V7 whose functions have been characterized in Rabdosia rubescens.
[0051] Figure 4 Verification diagram for the amplification of PlCYP706AA7 and PlCYP706AA14 gene fragments; M represents Marker, 1 and 2 both represent the fragments of PlCYP706AA7, and 3 and 4 both represent the fragments of PlCYP706AA14.
[0052] Figure 5 Bacterial inspection diagram of recombinant Escherichia coli strains (DH5α) of pYES2-PlCYP706AA7 and pYES2-PlCYP706AA14; M represents Marker, 1, 2, 3, and 4 all represent the bacterial inspection results of pYES2-PlCYP706AA7, and 5, 6, 7, and 8 all represent the bacterial inspection results of pYES2-PlCYP706AA14.
[0053] Figure 6 Colony PCR verification results of recombinant Saccharomyces cerevisiae strains (BY4742) of pYES2-PlCYP706AA7 / BY4742 and pYES2-PlCYP706AA14 / BY4742; M represents Marker, 1, 2, 3, and 4 all represent the bacterial inspection results of the recombinant strain pYES2-PlCYP706AA7 / BY4742, and 5, 6, 7, and 8 all represent the bacterial inspection results of the recombinant strain pYES2-PlCYP706AA14 / BY4742.
[0054] Figure 7 Schematic diagram of the reaction structure of paulownin catalyzed by microsomal proteins expressed by recombinant yeast strains of pYES2-PlCYP706AA7 / BY4742 and pYES2-PlCYP706AA14 / BY4742.
[0055] Figure 8 Liquid phase detection result diagram of the reaction of paulownin catalyzed by microsomal proteins expressed by recombinant yeast strains of pYES2-PlCYP706AA7 / BY4742 and pYES2-PlCYP706AA14 / BY4742. Specific implementation mode
[0056] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.
[0057] Example 1
[0058] Analysis of Chromosomal Location, Structure and Tissue Expression Patterns of Phryma leptostachya CYP706AA7 Gene and CYP706AA14 Gene
[0059] Combined with the Phryma leptostachya genome and transcriptomics data obtained in the early stage of the applicant's laboratory (NCBI accession number: PRJNA551634), and combined with the co-expression analysis of the known functional gene PlCYP81Q38 in the biosynthesis pathway of lignan compounds in three different tissues of Phryma leptostachya (root, stem and leaf), two Phryma leptostachya cytochrome 450 genes were screened out. They were respectively named CYP706AA7 (with the nucleotide sequence shown in SEQ ID NO: 1) and CYP706AA14 (with the nucleotide sequence shown in SEQ ID NO: 2) by the Plant CYP450 Nomenclature Committee. Both of these genes are located on chromosome 14 of Phryma leptostachya and are relatively close, as Figure 1 shown. The amino acid sequences were aligned using the DNAMAN software. The results showed that CYP706AA7 encodes 507 amino acids (as shown in SEQ ID NO: 3), CYP706AA14 encodes 518 amino acids (as shown in SEQ ID NO: 4), and the amino acid sequence identity between CYP706AA7 and CYP706AA14 is 71.29%. The amino acid sequences of CYP706AA7, CYP706AA14 and the characterized CYP450s (including the characterized SmCYP76AH1 and SmCYP76AH3 in Salvia miltiorrhiza, and the characterized IrCYP706V2 and IrCYP706V7 in Rabdosia rubescens) were aligned and visually analyzed using Clustal X and GeneDoc software, and the structures of Phryma leptostachya CYP706AA7 and CYP706AA14 were predicted using Alphafold3, as Figures 2 - 3 shown. From Figures 2 - 3It can be seen that the proteins encoded by the Phryma leptostachya CYP706AA7 gene and the CYP706AA14 gene both include a proline-rich region, a heme-binding region, an oxygen-binding and activation domain, and substrate recognition sites (SRS1-5), and have the typical structural characteristics of plant cytochrome P450 proteins.
[0060] Example 2
[0061] Construction and verification of recombinant expression vectors of Phryma leptostachya CYP706AA7 and CYP706AA14
[0062] The primers for amplifying the said fragments were designed using Primer5.0 and sent to Tsingke Company for primer synthesis (Table 1). The pYES2 vector was digested with BamH I and Hind III restriction endonucleases (the digestion program was: 37 °C, 30 min). Total RNA of Phryma leptostachya roots stored in an -80 °C refrigerator was taken, and Phryma leptostachya cDNA was reverse-transcribed according to the instructions of a reverse transcription kit (EG15133S, Jiangsu Yugong Biotechnology Co., Ltd.), and the CYP706AA7 and CYP706AA14 gene fragments were amplified using it as a template. The fragment amplification system was: 12.5 μL of 2× high-fidelity enzyme mixture, 1 μL of Phryma leptostachya cDNA, 0.5 μL of amplification primers F / R, 10.5 μL of sterile distilled water, with a total system of 25 μL, and 4 tubes were amplified. The amplification program was: 98 °C, 3 min; (98 °C, 10 s; 58 °C, 25 s; 72 °C, 1 min) for 35 cycles, and keep warm at 8 °C for 10 min. The PCR products were detected by agarose gel electrophoresis. As Figure 4 shown, the band sizes of the PCR products were consistent with the expectations, being 1554 bp and 1521 bp respectively. According to the steps of a column recovery kit (D6492020000J12X073, Omega, USA), the PCR products of the target genes were recovered. The recovered target fragments and the linearized vector were ligated using a homologous recombinase. The ligation system (10 μL) was: 2 μL of linearized vector, 1 μL of recovered target fragment, and 5 μL of homologous recombinase. The ligation condition was: 37 °C, 15 min.
[0063] The DH5α competent cells stored at -80°C were thawed on ice. The ligation product was added to the thawed competent cells. After placing on ice for 10 min, it was heat-shocked at 42°C for 50 s, immediately transferred to ice and placed for 3 min. 700 μL of the competent cells were added, and it was resuscitated on a shaker at 37°C for 30 min. After resuscitation, the supernatant was removed and spread on an LB resistant plate containing 100 μM ampicillin, and cultured overnight in an incubator at 37°C. Single colonies on the plate were picked into sterile water (8 single colonies were selected from each plate), and PCR detection was performed using the bacterial water as a template. The detection system was: 12.5 μL of 2× Taq Blue enzyme mixture, 1.5 μL of bacterial water, 0.5 μL of amplification primers pYES2-F / R, 10.5 μL of sterile distilled water, and the total system was 25 μL. The amplification program was: 95°C, 5 min; (95°C, 30 s; 58°C, 30 s; 72°C, 2 min) for 35 cycles, and incubated at 8°C for 10 min. The PCR products were detected by agarose gel electrophoresis, and the results were as Figure 5 shown. The band sizes of the PCR products were consistent with the expectations. Two of the correctly detected bacterial waters were selected and cultured in liquid LB medium containing 100 μM ampicillin, and sent to Tsingke Sequencing Company for sequencing. The correctly sequenced recombinant strains were selected for scale-up culture, and the recombinant plasmids were extracted with reference to the plasmid extraction kit (D6904030000H14X024, Omega, USA) and stored at -20°C for standby.
[0064] Table 1 PCR primer sequences
[0065]
[0066] Example 3
[0067] Transformation of the recombinant expression vectors of Phryma leptostachya CYP706AA7 and CYP706AA14 into Saccharomyces cerevisiae BY4742 and verification
[0068] The competent cells of Saccharomyces cerevisiae BY4742 stored in an -80°C refrigerator were thawed on ice. 500 ng of the correctly sequenced recombinant plasmid, 10 μL of salmon sperm DNA, 240 μL of filter-sterilized PEG3350, and 36 μL of sterile lithium acetate solution were added thereto. It was gently inverted and mixed evenly, placed at 30°C for 30 min, and placed at 42°C for 15 min. After transformation, it was centrifuged at 3000 r / min at room temperature for 1 min, the supernatant was removed, the cells were resuspended and washed with 1 mL of sterile water three times, and finally the cells were resuspended with 100 μL of sterile water and spread on an SC-Ura auxotrophic yeast medium plate and cultured at 30°C.
[0069] After 48 h of incubation, bacterial colonies grew. A yeast monoclonal was picked with a toothpick and placed in sterile water, and then subjected to cell wall breaking treatment by heating at 95 °C for 10 min in a PCR instrument. After the cell wall was broken, colony PCR was performed for detection. The detection system was as follows: 12.5 μL of 2× Taq Blue enzyme mixture, 1.5 μL of bacterial water, 0.5 μL of amplification primers pYES2-F / R, 10.5 μL of sterile distilled water, and the total system was 25 μL. The amplification program was: 95 °C, 5 min; (95 °C, 30 s; 58 °C, 30 s; 72 °C, 2 min) for 35 cycles, and incubation at 8 °C for 10 min. The electrophoresis result diagram of the PCR product is as shown in Figure 6 shown, indicating that the recombinant Saccharomyces cerevisiae strains pYES2-PlCYP706AA7 / BY4742 and pYES2-PlCYP706AA14 / BY4742 were successfully constructed.
[0070] Example 4
[0071] Induced expression and preparation of recombinant yeast microsomal proteins of Phryma leptostachya CYP706AA7 and CYP706AA14
[0072] A correctly detected positive clone was picked with a sterile toothpick and inoculated into 5 mL of SC-Ura defective yeast liquid medium containing 2% glucose, and cultured with shaking at 30 °C and 180 r / min for 24 h. 5 mL of the above bacterial liquid was inoculated into 100 mL of fresh SC-Ura defective yeast liquid medium containing 2% glucose, and cultured with shaking at 30 °C and 180 r / min for 24 h. At room temperature, centrifuged at 1500 g / min for 1 min, the supernatant was removed, and the cells were collected; then 30 mL of sterile water was added to wash the cells twice to remove the residual glucose on the cell surface. The washed cells were transferred to 100 mL of YPDA medium containing 2% galactose, and cultured with shaking at 30 °C and 180 r / min for 24 h to induce protein expression.
[0073] Centrifuge at 4℃, 1500g / min for 1min, remove the YPDA medium in the supernatant, and collect the bacteria; add 20mL of pre-cooled TEK solution to resuspend and wash the bacteria, place at room temperature for 5min, centrifuge at 4℃, 1500g / min for 1min, remove the supernatant; add pre-cooled TESB buffer to resuspend the bacteria (0.25g bacteria / mL). Pre-cool the low-temperature ultra-high pressure cell disruptor in advance, wait until it drops to 4℃, rinse the pipeline with TESB solution first, and after the pipeline is clean, set the pressure to 1850bar, add the yeast resuspended in TESB to disrupt, and after about 5 times of disruption, collect the yeast that has been disrupted and extract microsomal protein. The collected broken liquid was centrifuged at 4°C, 16500g / min for 15 min, the broken supernatant was collected and transferred to a new pre-cooled centrifuge tube, and two volumes of TESB buffer containing NaCl and PEG4000 were added thereto. After gently inverting to mix, it was allowed to stand on ice for 30 min, and then centrifuged at 4°C, 16500g / min for 15 min, the supernatant was removed, and TEG buffer was added to resuspend the microsomal protein, and stored at -80°C for later use.
[0074] Example 5
[0075] Enzyme activity reaction of substrates catalyzed by CYP706AA7 and CYP706AA14 microsomal proteins from Herba Lycopodii and detection of products
[0076] Weigh the paulownia tomentosa powder and prepare it into a 100mM mother liquor with DMSO for later use. The total reaction system is 300μL, which is as follows: 259μL of 100mM Tris-HCl buffer, 50μL of yeast microsomal protein encoded by the CYP706AA oxygen insertion enzyme gene of Herba Lycopodii, 0.6μL of NADPH, and 0.3μL of substrate paulownia tomentosa; the reaction conditions are 30℃ water bath for 2h, during which it can be gently inverted and mixed 2-3 times. After the reaction is completed, 1mL of ethyl acetate is added to terminate the reaction, and the product is extracted by ultrasonication for 10min. After centrifugation at 12000rpm / min for 3min at room temperature, the upper organic phase is aspirated and placed in a vacuum concentrator until the ethyl acetate is completely volatilized. 55% acetonitrile water is added to the residue in the centrifuge tube to resuspend it and dilute it to 200μL. The obtained sample solution is filtered with a 0.22μm filter for later use.
[0077] The reaction products were detected using a high performance liquid chromatography system. The chromatographic column was C 18 The column was 5 μm, 4.5×250 mm, the mobile phase was 55% acetonitrile water isocratic elution, the flow rate was 0.8 mL / min, the column temperature was 30°C, the detection wavelength was 285 nm, and the injection volume was 20 μL. Figures 7 - 8 As shown by Figure 7 It can be seen that inserting an oxygen atom between the C1 and C7 positions of Paulownia tomentosa can form the structure of thunbergii V.Figure 8 It can be seen that using the microsomal protein of pYES2 recombinant Saccharomyces cerevisiae as the negative control, the microsomal proteins of pYES2-PlCYP706AA7 / BY4742 and pYES2-PlCYP706AA14 / BY4742 recombinant Saccharomyces cerevisiae can both catalyze the formation of products from paulownin, and the position of the product peak is after the paulownin peak.
[0078] The above-described embodiments are some of the embodiments of the present invention, rather than all of the embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the present invention claimed, but rather represents selected embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art under the conditions of the concept of the present invention through relevant deductions and substitutions without creative efforts fall within the scope of protection of the present invention.
Claims
1. The CYP706AA oxygenase gene of Speranskia tuberculata, characterized in that, The Speranskia tuberculata CYP706AA oxygenase gene includes the CYP706AA7 gene and the CYP706AA14 gene, wherein, The CYP706AA7 gene: a1) Encodes the CYP706AA7 protein; a2) Has the nucleotide sequence shown in SEQ ID NO: 1; The CYP706AA14 gene: b1) Encodes the CYP706AA14 protein; b2) Has the nucleotide sequence shown in SEQ ID NO:
2.
2. Speranskia tuberculata CYP706AA oxygenase protein, characterized in that, The Speranskia tuberculata CYP706AA oxygenase protein includes the CYP706AA7 protein and the CYP706AA14 protein, wherein, The CYP706AA7 protein: A1) Has the amino acid sequence shown in SEQ ID NO: 3; A2) A derived amino acid sequence obtained by substituting, deleting, and / or adding one or several amino acids to the amino acid sequence in A1), and the derived amino acid sequence has the activity of the amino acid sequence shown in A1); or A3) An amino acid sequence having at least 80% homology with the amino acid sequence in A1); The CYP706AA14 protein: B1) Has the amino acid sequence shown in SEQ ID NO: 4; B2) A derived amino acid sequence obtained by substituting, deleting, and / or adding one or several amino acids to the amino acid sequence in B1), and the derived amino acid sequence has the activity of the amino acid sequence shown in B1); or B3) An amino acid sequence having at least 80% homology with the amino acid sequence in B1).
3. A recombinant expression plasmid containing the Speranskia tuberculata CYP706AA oxygenase gene as claimed in claim 1.
4. The recombinant expression plasmid according to claim 3, wherein The original plasmid used for the recombinant expression plasmid is the pYES2 plasmid.
5. The recombinant expression plasmid according to claim 4, characterized in that, The CYP706AA7 gene and the CYP706AA14 gene are inserted between the BamH I and Hind III sites of the original plasmid.
6. A recombinant strain expressing the Phryma leptostachya CYP706AA oxygenase gene as claimed in claim 1, characterized in that, The recombinant strain uses Saccharomyces cerevisiae BY4742 as the host bacterium and transforms the recombinant expression plasmid as claimed in claim 3 or 4.
7. Use of the Speranskia tuberculata CYP706AA oxygenase protein as claimed in claim 2 as a biocatalyst.
8. The application according to claim 7, wherein The application process of the Speranskia tuberculata CYP706AA oxygenase protein is: reacting in a reaction system containing a reaction substrate, NADPH, yeast microsomal protein encoded by the Speranskia tuberculata CYP706AA oxygenase gene, and Tris-HCl buffer.
9. The application according to claim 8, characterized in that, The reaction substrate is paulownin.
10. Use of the Speranskia tuberculata CYP706AA oxygenase protein as claimed in claim 2 in the oxygenation of paulownin to produce speranskialactone V.