Apigenin 8-hydroxylase protein and its encoding gene and application
By obtaining the 8-position hydroxytransferase sequence of apigenin from kale kesy and expressing recombinant proteins through eukaryotic expression, the problem of lack of isobaicin biosynthesis method was solved, and efficient biosynthesis of isobaicin was achieved, reducing costs and expanding the scope of application.
Patent Information
- Application Number
- CN202211085559.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-09-06
- Publication Date
- 2025-04-29
- Estimated Expiration
- 2042-09-06
AI Technical Summary
In the prior art, there is relatively lack of biological or chemical synthesis methods for isobaicin, and the chemical synthesis methods are costly and there are few reports of biosynthesis methods. It is difficult to fully study and develop the drug design and new compound generation potential of isobaicin.
By using the full-length transcriptome of the third generation of the Apigenin 8-position hydroxytransferase sequence was obtained and the recombinant protein was expressed by eukaryotic expression, confirming that LpCYP82D-1 could catalyze the production of isobaicin.
The biosynthesis of isobaicin has been achieved, the cost of chemical synthesis methods has been reduced, the scope of application of isobaicin has been expanded, and the experimental basis for its drug design and new compound generation is provided.
Smart Images
Figure CN115992106B_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of biotechnology, in particular to apigenin 8-hydroxylase protein and its encoding gene and application. Background Art
[0002] Isosculletarein is chemically named 5,7,8-trihydroxy-2-(4-hydroxyphenyl)chromen-4-one. Its molecular formula is C15H10O6, and it is soluble in chloroform, ethyl acetate, DMSO, etc. Isosculletarein is a class of 5,7,8-trihydroxyflavonoid compounds isolated from Scutellaria baicalensis, and is one of the main active ingredients of the genus Scutellariae, with a wide range of sources. At the same time, isosculletarein is also an important intermediate product for the synthesis of major flavonoid pharmacological ingredients in many plants. Through modification, rare natural compounds can be obtained, such as gardeniaflavin B that can induce human leukemia cell death, and scutellariae that is a selective inhibitor of human carboxylesterase 1 (hCE1) and clinically used for adjuvant treatment of tuberculosis. Pharmacological experimental studies have shown that isosculletarein has anti-tumor, anti-aging, antioxidant, and immune-enhancing effects. Takayuki et al. reported that baicalein has strong influenza virus inhibitory activity through in vivo and in vitro studies. Although baicalein is widely available, its expression level in natural plant species is low. Therefore, in order to fully study and develop the potential of baicalein for drug design and the generation of new natural or non-natural compounds, it is an important research task to obtain the synthesis method of baicalein and expand its application range.
[0003] At present, there are few reports on the synthesis of iso-scutellarin by biological or chemical modification at home and abroad. Halbwirth et al. first detected the activity of flavonoid 8-hydroxylase (F8H) from chrysanthemum petal microsomes. This activity was detected in the presence of NADPH and FAD. The enzyme can 8-hydroxylate flavonols and flavonoids. The Berim team isolated an 8-hydroxylase from basil, which exists in chloroplasts and belongs to the Rieske-type oxygenase protein family. It can convert tanshinone or onion yellow into 8-hydroxytanshinone or 8-hydroxyonion yellow, and is highly expressed in basil leaf trichomes. Zhao et al. isolated an 8-hydroxylase with high specificity for the substrate chrysin from Scutellaria baicalensis, which also has a weaker 6-hydroxylation (F6H) function. Subsequently, Gao et al. conducted convergent evolution studies on the CYP82D family in Scutellaria baicalensis and Erigeron breviscapus of the Lamiaceae family and the CYP706X family in the Asteraceae family, and reported five F8H functional enzymes unique to Scutellaria baicalensis that can catalyze apigenin to produce iso-scutellarin. Hu et al. reported a simplified chemical synthesis method for iso-scutellarin with a total yield of more than 50%. Summary of the invention
[0004] Although there are reports on the biological or chemical synthesis of iso-scutellarin, there are problems such as high cost of chemical synthesis methods and few reports on biological synthesis methods. The present invention is mainly based on the third-generation full-length transcriptome of the hanging stone moss, obtains the sequence of apigenin 8-hydroxytransferase, and confirms that LpCYP82D-1 can catalyze the production of iso-scutellarin through eukaryotic expression of recombinant protein.
[0005] The technical solution of the present invention is as follows:
[0006] The present invention provides a protein, which is the protein of a) or b) as follows:
[0007] a) a protein consisting of the amino acid sequence shown in Sequence 2 in the sequence listing;
[0008] b) A protein derived from a) having apigenin 8-hydroxylase activity, wherein one or more amino acid residues are substituted and / or deleted and / or added to the amino acid sequence shown in Sequence 2 in the sequence listing.
[0009] The apigenin 8-hydroxylase activity catalyzes the hydroxylation of the 8-position of the A ring of apigenin to generate isoscutellarin.
[0010] The coding gene of the protein also belongs to the protection scope of the present invention.
[0011] The coding gene is as shown in 1) or 2) or 3) below:
[0012] 1) Its nucleotide sequence is the DNA molecule shown in Sequence 1 in the sequence list;
[0013] 2) A DNA molecule that hybridizes with the DNA molecule defined in 1) under stringent conditions;
[0014] 3) A DNA molecule having 90% or more homology with the DNA molecule defined in 1) or 2).
[0015] The gene was named LpCYP82D-1, and the protein encoded by it was named LpCYP82D-1. The specific information is as follows: The sequence of the LpCYP82D-1 gene is shown in sequence 1 in the sequence list, which contains 1569 nucleotides and encodes the protein shown in sequence 2 in the sequence list, and sequence 2 consists of 522 amino acids.
[0016] Expression cassettes, recombinant expression vectors, transgenic cell lines or recombinant microorganisms containing the encoding gene also fall within the protection scope of the present invention.
[0017] The application of the protein as apigenin 8-hydroxylase also falls within the protection scope of the present invention.
[0018] The apigenin 8-hydroxylase is an enzyme that catalyzes the hydroxylation of the 8-position of the A ring of apigenin to generate isoscutellarin.
[0019] The use of the protein and the encoding gene in catalyzing the conversion of apigenin to produce isoscutellarin also falls within the protection scope of the present invention.
[0020] The invention is based on the third-generation and second-generation transcriptome data of Lysionotus pauciflorus, and uses a reverse genetics method to find and identify the specific hydroxylation modification enzyme LpCYP82D-1 for the synthesis of isobaicalein, thereby providing an apigenin 8-hydroxylase protein and its coding sequence and an experimental basis for the biosynthesis of isobaicalein. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] For purposes of illustration and not limitation, the present invention will now be described in terms of preferred embodiments thereof, with particular reference to the accompanying drawings, in which:
[0022] Figure 1 The expression levels of LpCYP82D-1 in various tissues of Chrysanthemum moss.
[0023] Figure 2 Verification results of pESC-His-LpCYPs vector transformation of WAT11.
[0024] Figure 3 UPLC diagram for the identification of the catalytic activity of LpCYP82D-1 on apigenin.
[0025] Figure 4 This is the MS identification diagram of the catalytic product of apigenin by LpCYP82D-1.
[0026] Figure 5 The molecular structures of apigenin (left) and isoscutellarin (right).
[0027] Figure 6 This is the conversion rate diagram of LpCYP82D-1 apigenin. DETAILED DESCRIPTION
[0028] Example 1: Apigenin 8-hydroxylase protein and its encoding gene and application
[0029] 1. Methods and steps for obtaining genes
[0030] Lysionotus pauciflorus (Non-patent literature on Lysionotus pauciflorus is: Huang Mei, Li Meijun, Huang Hong, et al. Species diversity and geographical distribution of wild Gesneriaceae in Guizhou Province [J]. Guangxi Plants, 2022, 42(2): 210-219. DOI: 10.11931 / guihaia.gxzw202008055.) Sample plants were collected from Xianheping Town, Anlong County, Guizhou Province, China. Four tissue samples of roots, stems, leaves, and flowers were quickly stored at -80℃ for multi-omics experiments. Based on the third-generation full-length transcriptome of Lysionotus pauciflorus, combined with the conserved domain of CYP450 and Nelson classification principles, 175 LpCYP450s sequences of 278aa-570aa A type were obtained. Through the joint analysis of the second-generation + third-generation transcriptome and the broad-target metabolome, LpCYP82D-1 was focused on.
[0031] The LpCYP82D-1 gene was synthesized and connected to the yeast expression vector pESC-His vector (the non-patent literature recording the yeast expression vector pESC-His vector is: Gao R, Lou Q, Hao L, Qi G, Tian Y, Pu X, He C, Wang Y, Xu W, Xu Z, Song J. Comparative genomics reveal the convergent evolution of CYP82D and CYP706X members related to flavone biosynthesis in Lamiaceae and Asteraceae. Plant J. 2022 Mar; 109(5): 1305-1318. doi: 10.1111 / tpj.15634. Epub 2021 Dec 27. PMID: 34907610.), and the connection sites were BamHI-F and SalI-R. The ligation system was directly transformed into TransT1 competent cells (purchased from Beijing Quanshijin Biotechnology Co., Ltd., product catalog number CB101-01), and positive clones were selected for sequencing (the total volume of the colony PCR system was 20 μL: 13 μL Mix Buffer, 1 μL template, 1 μL primer and 5 μL water, the program is as shown in Table 1, and the primers are as shown in Table 2), and compared with the transcript sequence. The nucleotide sequence had 100% similarity with the original transcriptome data.
[0032] Table 1 Colony PCR reaction procedure
[0033]
[0034] Table 2 Primer sequences used for colony PCR
[0035]
[0036] qRT-PCR was used to detect the true expression level of LpCYP82D-1 in tissues. The primers for the LpCYP82D-1 specific band of about 120 bp were designed, as shown in Table 3. Lp-Actin was used as the housekeeping gene, and the qPCR program was shown in Table 4. The roots, stems, leaves and flowers of Lysionotus pauciflorus were collected in Xianheping, Anlong County, Guizhou Province, and RNA was extracted. After adjusting the concentration to be consistent, it was reversely transcribed into cDNA for qPCR template. Finally, the expression levels of LpCYP82D-1 in the four tissues of Lysionotus pauciflorus were obtained, as shown in Table 3. Figure 1 The expression level of LpCYP82D-1 in flowers was significantly higher than that in roots, stems, and leaves, and the trend of transcriptome expression was consistent with that of qPCR expression.
[0037] Table 3 Primer sequences used for qRT-PCR
[0038]
[0039]
[0040] Table 4 qRT-PCR reaction procedure
[0041]
[0042] 2. Obtaining gene sequences and their encoded protein sequences
[0043] The obtained LpCYP82D-1 gene contains 1569 nucleotides, as shown in sequence 1 in the sequence list; it encodes a protein of 522 amino acids, as shown in sequence 2 in the sequence list, and the encoded protein is named LpCYP82D-1.
[0044] 3. Gene Function Verification
[0045] We used the eukaryotic system to verify the gene function. The pESC-His-LpCYPs vector was constructed previously. After sequencing confirmed that the sequence was correct, the vector was successfully transferred into the eukaryotic expression strain WAT11 (provided by Gao. The non-patent literature that records the eukaryotic expression strain WAT11 is: Gao R, Lou Q, Hao L, Qi G, Tian Y, Pu X, He C, Wang Y, Xu W, Xu Z, Song J. Comparative genomics reveal the convergent evolution of CYP82D and CYP706X members related to flavone biosynthesis in Lamiaceae and Asteraceae. Plant J. 2022 Mar; 109(5): 1305-1318. doi: 10.1111 / tpj.15634. Epub 2021 Dec 27. PMID: 34907610.) for in vivo verification in yeast.
[0046] The steps of LiCl yeast transformation are as follows:
[0047] 1. Take several yeast colonies with good growth on YPAD medium and place them in 5 mL YPAD liquid medium, and culture them at 30°C with shaking overnight;
[0048] 2. Take 1 mL of yeast solution and add it to 50 mL of YPAD medium. Continue to culture until its OD600 reaches 1.0;
[0049] 3. Centrifuge at 3000 g for 5 minutes to collect yeast cells;
[0050] 4. Add 20 mL of sterile distilled water for washing, centrifuge again at 3000 g for 5 minutes at room temperature, and discard the supernatant;
[0051] 5. Add 1 mL of sterile 0.1 mol / L LiCl solution to resuspend the cells, transfer to a 1.5 mL centrifuge tube, centrifuge at 12000 rpm for 15 seconds, and remove the supernatant;
[0052] 6. Resuspend the cells with 0.5-1 mL 0.1 mol / L LiCl and shake at 200 rpm at 30°C for 30 minutes;
[0053] 7. Take 5 μL salmon sperm DNA (1 μg / μL) from each transformation sample, heat denature at 94°C for 5 minutes, and then quickly cool on ice for 2 minutes;
[0054] 8. Aliquot yeast competent cells into 100 μL / tube, add 5 μL (about 1 μg) plasmid and 5 μL heat-denatured salmon sperm DNA, mix well. Then add 600 μL of PEG and LiCl mixed solution (final concentration is 40% PEG and 0.1 mol / L LiCl), vortex mix well;
[0055] 9. Heat shock in 30℃ water bath for 30 minutes, flicking to mix every 5 minutes; in 42℃ water bath for 15 minutes;
[0056] 10. Centrifuge at 12000 rpm for 15 seconds and remove the supernatant. Add 1 mL of sterile distilled water, vortex to mix, centrifuge at 12000 rpm for 15 seconds and remove the supernatant.
[0057] 11. Add 1 mL of sterile distilled water to suspend the cells and spread evenly on a yeast auxotrophic (-His) plate;
[0058] 12. Culture at 30℃ for 2-3 days and select single clones for PCR verification (such as Figure 2 ). Three LpCYP82D-1-pESC-His monoclones were selected, and all of them had positive bands at 1500 bp.
[0059] The induction, enzyme activity analysis and product identification of the recombinant protein are as follows:
[0060] 1) Induction of recombinant protein
[0061] LpCYP82D-1-pESC-His and pESC-His monoclonal colonies were picked up respectively and cultured in 2 mL SD-His liquid culture medium at 30° C. with shaking (200 rpm) for 2-3 days.
[0062] Take 0.1 mL of the cultured bacterial solution and add it to 20 mL of fresh SD-His culture medium (containing 2% glucose). After culturing at 30°C in a shaker (200 rpm) for 2-3 days, transfer the bacterial solution to a 50 mL sterile centrifuge tube, centrifuge at 1000 g for 10 min, collect the bacterial precipitate, and remove the supernatant. Resuspend the bacterial precipitate with 20 mL of sterile deionized water, centrifuge at 1000 g for 5 min, collect the bacterial precipitate, and remove the supernatant. Repeat 2-3 times.
[0063] Add 50 mL of SG-His liquid culture medium (containing 2% galactose) to resuspend the bacteria to an OD value of 1.0 at 600 nm, take 1 mL of the bacterial solution into a 15-mL sterile centrifuge tube, add apigenin at a final concentration of 100 μM (as shown in Table 5), and induce the reaction at 30°C shaking incubator (200 rpm) for 16 h.
[0064] 2) Determination of enzyme activity
[0065] Take 500 μL of bacterial solution for enzyme activity detection, terminate the reaction with three volumes of ethyl acetate, centrifuge at 13,000 rpm for 5 min, take the supernatant, concentrate at 45°C under low pressure for 1 h, add 200 μL of methanol to re-dissolve, and take 2 μL for sample loading.
[0066] Table 5 Recombinant protein yeast in vivo enzyme activity reaction system
[0067]
[0068] 3) Analysis and identification of enzyme activity products
[0069] Using the eukaryotic expression system, the recombinant protein of LpCYP82D-1 was successfully expressed, and further enzyme activity analysis was performed to identify their functions. The receptor of the enzyme activity reaction is apigenin. The UPLC spectrum of the enzyme activity product ( Figure 3 ), and found that LpCYP82D-1 was active against apigenin.
[0070] UPLC conditions:
[0071] UPLC model: Nexera UHPLC LC-20A system (SHIMADZU, Japan).
[0072] Mobile phase: Phase A: 0.1% formic acid aqueous solution; Phase B: acetonitrile.
[0073] Elution gradient: 0-7 min, 5%-100% B; 7-9 min, 100% B; 9-10.5 min, 100%-5% B; 10.5-11.5 min, 5% B.
[0074] DAD detection wavelength: 335nm.
[0075] Using UPLC to identify the enzyme activity product, it was found that there was only one peak of the enzyme activity reaction product of LpCYP82D-1 for apigenin as the receptor, and its retention time and UV spectrum were basically consistent with those of the standard substance isobaicalin. It was preliminarily determined that the product was isobaicalin ( Figure 3 ). The enzyme activity product was identified by mass spectrometry, and it was found that the MS information of product peak I was consistent with that of the standard substance isoscutellarin, and the main ion fragments were 285, 213 and 117 ( Figure 4 ). In summary, the in vitro enzyme activity evidence showed that LpCYP82D-1 encodes a hydroxytransferase that catalyzes the 8-hydroxylation of apigenin to produce isoscutellarin. Figure 5 The molecular structures of apigenin (left, substrate) and baicalein (right, product). Baicalein is 8-hydroxyapigenin. LpCYP82D-1 can transfer the hydroxyl group to the 8th position of the A ring of apigenin to generate baicalein.
[0076] Mass spectrometry conditions:
[0077] Sample preparation before mass spectrometry is the same as sample preparation before UPLC.
[0078] The samples were separated by UPLC MS / MS using an InfinityLab Poroshell 120EC-C18 column (2.1*50mm 1.9-micron; Agilent). The mobile phase was the same as that of UPLC. The elution gradient was 0 min, 5% B; 7 min, 95% B, and finally 95% B equilibrium (7-8 min). The flow rate was 0.30 mL / min, and the detection wavelength was the same as above.
[0079] UPLC MS / MS mass spectrometry conditions, electrospray ionization, full ion scan, negative-ion (EI) mass spectrometry analysis. The negative ion mode conditions are as follows: gas temperature: 300℃, gas flow: 6L / min, nebulizer: 30psi, sheath gas temperature: 300℃, sheath gas flow: 11L / min, capillary voltage: 2500V, nozzle voltage: 2000V, collision energy: 30V. The characteristic ions of baicalein in negative ion mode are 285.1, 213.1 and 117.1.
[0080] 4) Catalytic activity of LpCYP82D-1
[0081] To further understand the catalytic properties of LpCYP82D-1 recombinant protein, we tested the catalytic activity of LpCYP82D-1 for apigenin. The enzyme activity reaction system was the same as 2). The substrate conversion rate (%) was used as the catalytic activity comparison parameter, and the calculation formula = product peak area / (product peak area + substrate peak area)*100%. The experiment found that the efficiency of LpCYP82D-1 recombinant protein in converting apigenin to iso-scutellarin was higher than 50% (e.g. Figure 6 ), indicating that the recombinant protein LpCYP82D-1 has high catalytic efficiency for apigenin.
[0082] The above specific implementations do not constitute a limitation on the protection scope of the present invention. It should be understood by those skilled in the art that various modifications, combinations, sub-combinations and substitutions may occur depending on design requirements and other factors. Any modification, equivalent substitution and improvement made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.
Claims
1. A protein, the amino acid sequence of which is shown in SEQ ID NO:
2.
2. The gene encoding the protein according to claim 1, characterized in that: The nucleotide sequence of the encoding gene is shown in SEQ ID NO:
1.
3. An expression cassette, recombinant expression vector, transgenic cell line or recombinant microorganism containing the encoding gene according to claim 2.
4. The protein according to claim 1 is used in catalyzing the conversion of apigenin to isoscutellarin.
5. The coding gene according to claim 2 is used for catalyzing the conversion of apigenin to isoscutellarin.