Apigenin 6-hydroxylase protein and its encoding gene and application

Abstract

The present invention discloses apigenin 6-hydroxylase protein and its encoding gene and application. The apigenin 6-hydroxylase protein provided by the present invention is a protein of a) or b) as follows: a) a protein composed of the amino acid sequence shown in sequence 2 in the sequence table; b) a protein derived from a) with apigenin 6-hydroxylase activity, in which 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 table. The present 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‑8 synthesized by baicalein, providing apigenin 6-hydroxylase protein and its encoding sequence and experimental basis for the biosynthesis of baicalein.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to apigenin 6-hydroxylase protein and its encoding gene and application. Background Art

[0002] Scutellarin is chemically named 5,6,7-trihydroxy-2-(4-hydroxyphenyl)chromen-4-one. Its molecular formula is C15H10O6 and it is soluble in DMSO and the like. Scutellarin is a compound isolated from Scutellaria baicalensis and is one of the main active ingredients of Scutellaria plants. It has a wide range of sources. At the same time, scutellarin is also an important intermediate product for the synthesis of major flavonoids in many plants. Rare natural compounds can be obtained through modification, such as gardeniaflavin B, which can induce death of human leukemia cells, caryophyllin, which has neuroprotective, anti-tumor, cytotoxic and immunomodulatory effects, and scutellarin, which is a selective inhibitor of human carboxylesterase 1 (hCE1) and is clinically used for adjuvant treatment of tuberculosis. Pharmacological experimental studies have shown that baicalin has liver protection, anti-tumor, antibacterial and antiviral, antioxidant activities and effective activity against neurodegenerative diseases. It has cytotoxic activity against human leukemia cells, inhibitory activity against 17β-HSD and human salivary α-amylase, and it is an antagonist of tromboxane A2 receptor, as one of the formulations of new drugs involved in thrombotic diseases. The latest pharmacological studies have shown that baicalin can inhibit SARS coronavirus helicase and has been used in traditional Chinese medicine to treat inflammatory diseases such as COVID-19. Based on the good biological activity and clinical needs of baicalin, it is an important research work to explore the efficient synthesis method of baicalin and expand its application range.

[0003] There have been many research reports on the synthesis of baicalin by structural modification at home and abroad. At present, the main feasible ways to modify the structure of baicalin are chemical synthesis and biosynthesis. Cui et al. used 2-acetyl-3,4,5-trimethoxyphenol as raw material and first proposed a three-step reaction to synthesize baicalin. Subsequently, Righi et al. optimized the chemical synthesis route of baicalin, using naringenin as raw material to obtain baicalin (total yield 62%) and its methylated derivatives. Liu Zhihui et al. transformed baicalin by microbial fermentation, screened out Aspergillus niger with strong ability to transform baicalin from 28 strains, and then isolated baicalin from the fermentation broth. Ju Wenzheng et al. studied the transformation of baicalin in hydrochloric acid, β-glucuronidase and intestinal flora, and found that it can be converted into aglycone form. The use of chemical methods for structural modification has the disadvantages of low selectivity, many by-products, difficulty in subsequent separation, large equipment investment, high technical requirements, large use of organic solvents, and potential environmental pollution. The use of hydrolysis conversion and microbial conversion methods to obtain scutellariae also has the disadvantages of low selectivity and high technical requirements. Summary of the invention

[0004] Although there are reports on the biological or chemical synthesis of scutellarin, these methods have problems such as being environmentally unfriendly, having a diversity of target products, and having high preparation conditions. The present invention is mainly based on the third-generation full-length transcriptome of the apigenin 6-hydroxytransferase sequence obtained, and through eukaryotic expression of the recombinant protein, confirmed that LpCYP82D-8 can catalyze the production of scutellarin.

[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 6-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 6-hydroxylase activity catalyzes the hydroxylation of the 6-position of the A ring of apigenin to generate baicalein.

[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-8, and the protein encoded by it was named LpCYP82D-8. The specific information is as follows: The LpCYP82D-8 gene sequence is shown in sequence 1 in the sequence list, which contains 1578 nucleotides and encodes the protein shown in sequence 2 in the sequence list, and sequence 2 consists of 525 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 6-hydroxylase also falls within the protection scope of the present invention.

[0018] The apigenin 6-hydroxylase is an enzyme that catalyzes the hydroxylation of the 6-position of the A ring of apigenin to generate baicalein.

[0019] The application of the protein and the encoding gene in catalyzing the conversion of apigenin to baicalein 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-8 for the synthesis of baicalein, thereby providing the 6-hydroxylase protein of apigenin and its coding sequence and experimental basis for the biosynthesis of baicalein. 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-8 in various tissues of Chrysanthemum moss.

[0023] Figure 2 Verification results of pESC-His-LpCYPs vector transformation of WAT11.

[0024] Figure 3 UPLC chart for the identification of the catalytic activity of LpCYP82D-8 on apigenin.

[0025] Figure 4 This is the MS identification diagram of the product catalyzed by LpCYP82D-8 on apigenin.

[0026] Figure 5 The molecular structures of apigenin (left) and baicalin (right)

[0027] Figure 6 This is the apigenin conversion rate diagram of LpCYP82D-8 and SbCYP82D1.1. DETAILED DESCRIPTION

[0028] Example 1: Apigenin 6-hydroxylase protein and its encoding gene and application

[0029] one, 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-8 was focused on.

[0031] The LpCYP82D-8 gene was synthesized and connected to the yeast expression vector pESC-His vector (provided by Gao, 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 shown in Table 1, and the primer sequences used for colony PCR are shown in Table 2), and compared with the transcript sequence, the nucleotide sequence was 100% similar to the original data. The reported SbCYP82D1.1(F6H) (Gao R, Lou Q, Hao L, Qi G, Tian Y, Pu X, HeC, Wang Y, Xu W, Xu Z, Song J. Comparative genomics reveal the convergentevolution of CYP82Dand CYP706X members related to flavone biosynthesis in Lamiaceae and Asteraceae. Plant J. 2022Mar;109(5):1305-1318.doi:10.1111 / tpj.15634.Epub 2021Dec 27. PMID:34907610.) was used as the positive control, and the nucleotide sequence had a similarity of 50.91% with the positive control sequence.

[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-8 in tissues. The primers for the LpCYP82D-8 specific band of about 150 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-8 in the four tissues of Lysionotus pauciflorus were obtained, as shown in Table 3. Figure 1 The expression level of LpCYP82D-8 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] Table 4 qRT-PCR reaction procedure

[0040]

[0041] 2. Obtaining gene sequences and their encoded protein sequences

[0042] The obtained LpCYP82D-8 gene contains 1578 nucleotides, as shown in sequence 1 in the sequence list; it encodes a protein of 525 amino acids, as shown in sequence 2 in the sequence list, and the encoded protein is named LpCYP82D-8.

[0043] 3. Gene Function Verification

[0044] We used the eukaryotic system to verify the gene function. The pESC-His-LpCYPs vector was constructed in the previous article. 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 recorded 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. The specific transformation method is as follows:

[0045] 1) Preparation of competent yeast

[0046] Take several yeast colonies with good growth on YPAD medium and add them to 5 mL YPAD liquid medium. Incubate at 30°C overnight with shaking. Take 1 mL of yeast liquid and add it to 50 mL YPAD medium. Continue to culture until the OD 600 to 1.0.

[0047] Collect yeast cells by centrifugation at 3000g for 5 minutes, add 20mL of sterile distilled water to wash, centrifuge again at 3000g for 5 minutes at room temperature, and discard the supernatant. Add 1mL of sterile 0.1mol / L LiCl solution to resuspend the cells, transfer to a 1.5mL centrifuge tube, centrifuge at 12000rpm for 15 seconds, and discard the supernatant. Resuspend the cells with 0.5-1mL 0.1mol / LLiCl, shake at 30℃, 200rpm for 30 minutes, take 5μL of salmon sperm DNA (1μg / μL) for each transformation sample, heat denature at 94℃ for 5 minutes, and then quickly put on ice to cool for 2 minutes.

[0048] 2) LiCl yeast transformation

[0049] Aliquot yeast competent cells into 100 μL / tube, add 5 μL (about 1 μg) plasmid and 5 μL heat-denatured salmon sperm DNA, and mix well. Then add 600 μL of PEG and LiCl mixed solution (final concentration is 40% PEG and 0.1 mol / L LiCl), and vortex to mix well. Heat shock in a 30°C water bath for 30 minutes (gently flick to mix every 5 minutes), and then bathe in a 42°C water bath for 15 minutes. Centrifuge at 12000 rpm for 15 seconds and remove the supernatant. Add 1 mL of sterile distilled water, vortex to mix well, centrifuge at 12000 rpm for 15 seconds, and remove the supernatant. Add 1 mL of sterile distilled water to suspend the cells and evenly spread on a yeast nutrient-deficient (-His) plate.

[0050] The plate was incubated at 30°C for 2-3 days, and a single clone was selected for PCR verification (e.g. Figure 2 ). Three LpCYP82D-8-pESC-His monoclones were selected, and all of them had positive bands at 1500 bp.

[0051] The induction, enzyme activity analysis and product identification of the recombinant protein are as follows:

[0052] 1) Induction of recombinant protein

[0053] LpCYP82D-8-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.

[0054] 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.

[0055] 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, place in a new Erlenmeyer flask, 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 and react at 30°C shaking incubator (200 rpm) for 16 h.

[0056] 2) Determination of enzyme activity

[0057] Take 500 μL of the reaction solution for enzyme activity detection. Use three times the volume of ethyl acetate to terminate the reaction, 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. Take 2 μL for sample loading.

[0058] Table 5 Recombinant protein yeast in vivo enzyme activity reaction system

[0059]

[0060] 3) Analysis and identification of enzyme activity products

[0061] Using the eukaryotic expression system, the recombinant protein of LpCYP82D-8 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-8 was active against apigenin.

[0062] UPLC conditions:

[0063] UPLC model: Nexera UHPLC LC-20A system (SHIMADZU, Japan).

[0064] Mobile phase: Phase A: 0.1% formic acid aqueous solution; Phase B: acetonitrile.

[0065] 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.

[0066] DAD detection wavelength: 335nm.

[0067] 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-8 for apigenin as the receptor, and its retention time and UV spectrum were basically consistent with those of the standard scutellarin. It was preliminarily determined that the product was scutellarin ( 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 scutellariae, and the main ion fragments were 285 and 117 ( Figure 4 ). In summary, the in vitro enzyme activity evidence showed that LpCYP82D-8 encodes a hydroxytransferase that catalyzes the 6-hydroxylation of apigenin to baicalein. Figure 5 The molecular structures of apigenin (left, substrate) and baicalein (right, product). Baicalein is 6-hydroxyapigenin.

[0068] Mass spectrometry conditions:

[0069] Sample preparation before mass spectrometry is the same as sample preparation before UPLC.

[0070] 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.

[0071] 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 scutellariae in negative ion mode are 285.0 and 117.0.

[0072] 4) Catalytic activity of LpCYP82D-8

[0073] In order to further understand the catalytic properties of the LpCYP82D-8 recombinant protein, we used the reported SbCYP82D1.1 as a positive control to detect the catalytic activity of SbCYP82D1.1 and LpCYP82D-8 for apigenin. The enzyme activity reaction system is the same as 2). The substrate conversion rate (%) is 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 the LpCYP82D-8 recombinant protein in converting apigenin to baicalein is close to 60%, which is about twice the conversion rate of SbCYP82D1.1, indicating that the recombinant protein LpCYP82D-8 has a higher catalytic efficiency for apigenin (such as Figure 6 ).

[0074] 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 into baicalein.

5. The coding gene according to claim 2 is used for catalyzing the conversion of apigenin into baicalein.

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