Cnidium monnieri hydroxylase gene CmC2′H and its application

Abstract

The invention discloses the application of hydroxylase encoded by Cnidium monnieri CmC2'H gene in biosynthesis of coumarin compounds, belonging to the field of biotechnology. The nucleotide sequence of CmC2'H gene is shown in SEQ ID NO.1, or is completely complementary to the sequence shown in SEQ ID NO.1, or is a nucleotide sequence encoding an amino acid sequence shown in SEQ ID NO.2. Umbelliferone is obtained by an enzyme-catalyzed biosynthesis method using CmC2'H protein in vitro, providing a new method for synthesizing umbelliferone.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to cnidium monnieri hydroxylase and application thereof. Background Art

[0002] In recent years, the rise of molecular biology and metabolic engineering has brought new ideas and methods to people. Its basic idea is to use metabolic engineering methods to produce active pharmaceutical ingredients at the level of organisms or organelles (such as Escherichia coli and yeast). At present, the development of this technology is mainly limited by the difficulty in finding key proteases and genes encoding them for synthesizing active pharmaceutical ingredients. However, this method has the advantages of less time consumption, high unit yield, and no influence from natural conditions such as seasons and climate. Therefore, the use of molecular biology and metabolic engineering technology to produce active pharmaceutical ingredients has broad application prospects.

[0003] Coumarin compounds are the main compounds in Cnidium monnieri and are a class of secondary metabolites. Structurally, coumarins usually have a 1,2-benzopyrone scaffold and are generally divided into three categories: simple coumarins, furanocoumarins, and pyranocoumarins. Although the structural skeleton of coumarins is not complex, their biosynthetic mechanism is still unclear. Among the coumarins, umbelliferone plays a vital role in the plant phenylpropanoid metabolic pathway and is one of the hubs of the biosynthetic pathway of coumarin compounds. Umbelliferone is a key intermediate in the biosynthesis of isoprenylcoumarins, from which furanocoumarins and pyranocoumarins are derived, such as psoralen and seselin. When umbelliferone reacts with the isoprenyl donor substrate dimethylallyl pyrophosphate (DMAPP), umbelliferone forms 7-Demethylsuberosin (DMS) and osthenol under the catalysis of 6-Prenyltransferase (6-PT) and 8-Prenyltransferase (8-PT), respectively. This step is the entry point of the furanocoumarin biosynthesis pathway, which can form linear and angular furanocoumarins. However, to date, the hydroxylation mechanism of different types of coumarins and the mechanism of plant evolution to produce different types of coumarins remain unclear. Therefore, obtaining new hydroxylation genes from Cnidium monnieri and conducting functional verification can open up new ways to produce complex coumarin compounds using biosynthetic methods and to regulate the quality of Cnidium monnieri using plant genetic engineering methods. Summary of the invention

[0004] The present invention mainly aims at the above technical problems and provides a hydroxylase gene for catalyzing the production of umbelliferone and a method for biosynthesizing umbelliferone to solve the shortcomings of the existing chemical synthesis of umbelliferone.

[0005] Specifically, the present invention provides the following technical solutions:

[0006] In one aspect, the present invention provides a hydroxylase, whose amino acid sequence comprises the sequence shown in SEQ ID NO.2.

[0007] In another aspect, the present invention provides a hydroxylase gene, the sequence of which is a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO.2.

[0008] Or based on the principle of complementary pairing, the hydroxylase gene provided by the present invention can be a sequence that is fully complementary to the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.2.

[0009] In a preferred embodiment, the nucleotide sequence of the hydroxylase gene comprises a nucleotide sequence as shown in SEQ ID NO: 1, or a nucleotide sequence that is completely complementary to the sequence shown in SEQ ID NO. 1.

[0010] It is well known to those skilled in the art that a gene sequence may also contain introns, promoters and various regulatory elements, and thus the nucleotide sequence of the hydroxylase gene may also contain introns, promoters and various regulatory elements.

[0011] In another aspect, the present invention provides a recombinant expression vector encoding the aforementioned hydroxylase.

[0012] In another aspect, the present invention provides a transgenic recombinant bacterium encoding the aforementioned hydroxylase.

[0013] On the other hand, the present invention provides an application of a hydroxylase gene in the synthesis of umbelliferone, wherein the nucleic acid sequence of the hydroxylase gene is a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO.2, or a sequence that is fully complementary to a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO.2.

[0014] In a preferred embodiment, the hydroxylase gene sequence is a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.2, or a sequence that is completely complementary to the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.2.

[0015] In a preferred embodiment, the nucleotide sequence of the hydroxylase gene is as shown in SEQ ID NO: 1, or a nucleotide sequence that is completely complementary to the sequence shown in SEQ ID NO. 1.

[0016] In another aspect, the present invention provides use of the aforementioned hydroxylase in synthesizing coumarin compounds.

[0017] In another aspect, the present invention provides a method for synthesizing umbelliferone, the method comprising the following steps:

[0018] 1) obtaining the aforementioned hydroxylase;

[0019] 2) Using the hydroxylase in step 1) to catalyze the synthesis of umbelliferone in an enzyme activity reaction system.

[0020] In a preferred embodiment, the hydroxylase is obtained by prokaryotic expression.

[0021] In a preferred embodiment, the hydroxylase is obtained by chemical synthesis.

[0022] In a preferred embodiment, the enzyme activity reaction system contains the above-mentioned hydroxylase, α-ketoglutarate, vitamin C, FeSO4, and buffer.

[0023] In another aspect, the present invention provides a method for synthesizing coumarin compounds, characterized in that the method comprises the following steps:

[0024] 1) obtaining a hydroxylase comprising the amino acid sequence shown in SEQ ID NO.2;

[0025] 2) Using the above hydroxylase to catalyze the synthesis of umbelliferone in an enzyme activity reaction system.

[0026] 3) Umbelliferone in step 2) is further used as a substrate for synthesizing coumarin compounds.

[0027] In a preferred embodiment, the hydroxylase is obtained by prokaryotic expression.

[0028] In a preferred embodiment, the hydroxylase is obtained by chemical synthesis.

[0029] In a preferred embodiment, the enzyme activity reaction system contains the above-mentioned hydroxylase, α-ketoglutarate, vitamin C, FeSO4, and buffer.

[0030] In a preferred embodiment, the reaction system of step 3) further contains other necessary components known in the art for synthesizing coumarin compounds.

[0031] Compared with the prior art, the present invention has the following advantages:

[0032] 1) The present invention provides a novel hydroxylase.

[0033] 2) Compared with the prior art, the present invention uses an enzyme-catalyzed biosynthesis method to obtain umbelliferone, which has the advantages of simpler steps, less pollution, and a more uniform product compared to chemical synthesis.

[0034] 3) The substrate generated by the hydroxylase in the present invention can be used as a synthetic substrate for coumarin compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The method of the present invention and its beneficial effects are described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0036] Figure 1 This is the SDS-PAGE purity test chart of the protein after nickel column affinity chromatography purification (M: Protein Maker; 1: pET28a-CmC2'H protein purification solution)

[0037] Figure 2 Schematic diagram of the hydroxylation of coumaroyl-CoA catalyzed by CmC2'H protein to synthesize umbelliferone.

[0038] Figure 3 This is a mass spectrum of the product catalyzed by CmC2'H protease detected by mass spectrometry in Example 2, including: a mass spectrum detection diagram of the standard umbelliferone and a mass spectrum detection diagram of the sample catalyzed by CmC2'H protein. DETAILED DESCRIPTION

[0039] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by ordinary technicians in the technical field to which this application belongs.

[0041] Example 1

[0042] Cloning of target hydroxylase gene

[0043] According to the previous analysis of the full-length transcriptome sequencing results of Cnidium monnieri, a pair of specific primers (CmC2'HF: 5'-GTGCCGCGCGGCAGCCATATGGCTCCATCTACCAC-3' and CmC2'HR: 5'-GTGGTGGTGGTGGTGCTCGAGTCACATCTTTGCGAAATCAAG-3') were designed. Total RNA was extracted from Cnidium monnieri leaves using AG RNAex Pro RNA extraction reagent and SteadyPure RNA extraction kit, and reverse transcribed to synthesize cDNA. The CDS sequence of the CmC2'H gene in Cnidium monnieri that catalyzes p-coumaroyl-CoA-2'-hydroxylation as shown in SEQ ID NO: 1 was amplified using the above primers from the cDNA obtained by reverse transcription. The full length of the CDS sequence of the CmC2'H gene is 1053 bp.

[0044] The specific steps for obtaining the hydroxylase gene CmC2'H are as follows:

[0045] (1) Grind fresh Cnidium monnieri leaves with liquid nitrogen cooling, accurately weigh 100 mg of powder and add it to a 2 mL centrifuge tube pre-cooled with liquid nitrogen, then add AG RNAex Pro RNA extraction reagent, vortex mix and let stand for 10 min;

[0046] (2) Total RNA was extracted from leaves of Cnidium monnieri according to the instructions of the SteadyPure RNA extraction kit;

[0047] (3) Using the total RNA extracted from the leaves of Cnidium monnieri as a template, the reverse transcriptase kit PrimeScript TM IV1st strand cDNA Synthesis Mix (purchased from Beijing Bio-Rad Biotechnology Co., Ltd.) was used to reverse transcribe and synthesize the first strand of cDNA. The reaction conditions were carried out according to the instructions of the kit;

[0048] (4) Using the above primers CmC2'HF and CmC2'HR and Hieff Gold High Fidelity DNA Polymerase (purchased from Shanghai Yisheng Biotechnology Co., Ltd.) was used to amplify the CDS sequence of the CmC2'H gene in Cnidium monnieri that catalyzes the 2'-hydroxylation of p-coumaroyl-CoA from the cDNA obtained by reverse transcription of RNA. The reaction product was purified using an agarose gel DNA recovery kit.

[0049] (5) Reaction conditions:

[0050] 95℃ pre-denaturation for 3min; 95℃15sec, 55℃10sec, 72℃45sec, 30 cycles; 72℃ extension for 5min. The purified PCR product was inserted between the XhoI and NdeI restriction sites of the expression vector pET28a using ClonExpress IIOne Step Cloning Kit (purchased from Nanjing Novogene Biotechnology Co., Ltd.), transformed into Escherichia coli DH5α competent cells, screened positive transformants and sequenced to obtain the required full-length gene. Plasmids carrying the CmC2'H gene sequence were extracted from the positive transformants and named pET28a-CmC2'H plasmids.

[0051] The nucleotide sequence and the encoded amino acid sequence obtained by sequencing are shown as SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0052] SEQ ID NO.1

[0053]

[0054] SEQ ID NO.2

[0055] MAPSTTDDYRHFAVTKGHGVKGLSDLQLGALPEQYIQPVEERLDMTKVLKEESIPVIDMSNLDDPKVAEQIAAAAEKWGFFQIVNHGVPIEVLENVQEATRRFFALPVEEKIKFTQEHSPTNSVRLTTSFLPKIDKVLEWKDYLSILVSDKNSSEFWPSSCKKEVMEYVEKSEFV MKWLLKALMQGLNVDMDSKESLLMGSTRINLNYYPVCPNPELAIGVGRHSDVSSLTFLLQDNVGGLHIRKMETDDTWIYVPPITGAIVINIGDALQILSNGKYKSAEHCVAANGSHNRISVPIFANPSPDDIIGPLPEVLKNGEKPIYKHVLYSDYVKHFYRKSHDGKDTLDFAKM

[0056] The amino acid sequence obtained by sequencing was compared with the closest sequences on NCBI (KY445967, MK350251), and it was found that there were multiple amino acid mutations inside the DIOX_N domain (pfam14226) and on the flank (right side) of the domain, which may affect the activity of the enzyme.

[0057] Example 2 Induced expression and purification of CmC2'H protein

[0058] In order to analyze the catalytic function of the hydroxylation gene CmC2'H, the recombinant strain obtained in Example 1 was recombinantly expressed in Escherichia coli BL21, and its gene function was verified by feeding the precursor compound coumaroyl-CoA of the gene catalytic reaction and detecting the synthesis of the product by mass spectrometry.

[0059] The specific steps are as follows:

[0060] (1) The pET28a-CmC2'H plasmid obtained in Example 1 was introduced into Escherichia coli BL21; a blank vector was used as a negative control and transformed into Escherichia coli BL21.

[0061] (2) Spread the transformed E. coli BL21 on LB solid medium containing Kan antibiotics, place the plate upside down in a 37°C incubator overnight, and grow a single clone.

[0062] (3) Single clones with a diameter of about 1-2 mm were selected and placed in LB liquid medium containing Kan antibiotics. The culture was shaken at 37°C and 200 rpm, and positive transformants were verified by bacterial liquid PCR.

[0063] (4) Take 100 μl of positive cloned bacterial solution and transfer it to 500 mL of LB liquid medium containing Kan antibiotics, and culture it at 37°C and 200 rpm for 12-16 h until OD600 reaches about 0.5, then add 0.5 mM IPTG and induce it at 25°C and 120 rpm for 12-16 h.

[0064] (5) The induced BL21 bacterial solution was centrifuged at 4000 rpm for 15 min to collect the cells, the supernatant was discarded, and the cells were resuspended in 10 mL of 4°C precooled Tris HCl buffer (pH 6.5). Then 100 μM PMSF was added and resuspended. The resuspended cells were ultrasonically disrupted in an ice water bath for 10 min until the bacterial solution was basically clear. The cells were centrifuged at 4°C and 10,000 rpm for 10 min. The supernatant was the soluble protein crude enzyme solution.

[0065] (6) Protein purification was performed using the Ni-protein purification gravity column of Shanghai Shenggong Co., Ltd. The supernatant of the bacterial solution after crushing and centrifugation was first filtered with a 0.22 μm filter membrane and then used for column purification. First, the nickel column was activated with 3 times of 5 mL of permeabilized water, and then 3-5 times of 5 mL of Lysis buffer (20 mM imidazole, 20 mM HEPES, 500 mM NaCl, 10% glycerol) was added to balance the nickel column. After that, the filtered protein sample was loaded onto the column and incubated on ice for 10 minutes. The flow-through liquid could be collected at the bottom of the tube as required. After the sample loading was completed, Lysis buffer was added 3 times to remove some impurities adsorbed on the column. Finally, 5 mL of Elution buffer (300 mM imidazole, 20 mM HEPES, 500 mM NaCl, 10% glycerol) was added. The filtrate was the required protein purification solution (pure enzyme solution), and the concentration of the purified protein was determined by ultraviolet spectrophotometer.

[0066] (7) According to the instructions of the SDS-PAGE gel preparation kit (purchased from Wuhan Servicebio Biotechnology Co., Ltd.), first prepare 8% separation gel. According to the experimental requirements, mix solutions A and B in equal proportions, add an appropriate amount of improved coagulant, and prepare the lower gel solution and the upper gel solution respectively. The preparation volume of the upper gel and lower gel solutions can be adjusted in equal proportion according to the glass plate with a thickness of 1.0 mm.

[0067] (8) Assemble the gel preparation tool, add the prepared lower gel solution first, then use distilled water or ethanol to seal the lower gel surface, wait for the lower gel to solidify fully (about 10-15 minutes), discard the water or ethanol, and use filter paper to absorb the remaining water or ethanol, then add the prepared upper gel solution, insert the comb, and wait for it to solidify (about 10-15 minutes) before use; you can also add the prepared upper gel solution slowly and evenly immediately after adding the prepared lower gel solution, insert the comb, and wait for it to solidify (about 10-15 minutes) before use. Electrophoresis conditions: Use Tris-Glycine electrophoresis buffer (G2018) for electrophoresis: upper gel 90V, about 30 minutes (marker enters the separation gel); lower gel 150-180V, about 60-90 minutes. Staining: Carefully remove the gel from the glass plate with a gel plate, place it in a plate containing an appropriate amount of rapid staining solution, and stain for 30 minutes at 25°C and 45rpm. Place the above protein gel under a gel imaging system to observe the protein electrophoresis results.

[0068] The results show that Figure 1 ), and after purification by nickel column affinity chromatography, a relatively pure pET28a-CmC2'H protein solution was obtained.

[0069] Example 3 In vitro enzyme activity detection

[0070] 1. Enzyme activity reaction system: Reaction volume 200μL: 10mM p-coumaryl-CoA 3.0μL; 0.2M α-ketoglutaric acid 0.5μL; 100mg / mL vitamin C 0.5μL; 0.5M FeSO4 0.5μL; 2μg purified protein (CmC2'H), 100mM Tris-HCl buffer (pH 6.5) to 200μL. React at 20℃ for 5h, and add 100μL methanol to terminate the reaction. At the same time, in order to determine the kinetic parameters, p-coumaryl-CoA was added at concentrations ranging from 10 to 200 μM, and the Km (Maxwell-Boltzmann constant) and Kcat (catalytic constant) values ​​were compared with those of two reported p-coumaryl-CoA-2′-hydroxylases, PpC2′H (Genbank accession numbers KY445967) and AdC2′H (Genbank accessionnumbers MK350251).

[0071] 2. Liquid chromatography parameters: chromatographic column: Agilent Zorbax SB-C18 (250 mm×4.6 mm, 5 μm); column temperature: 30°C; flow rate: 1.0 mL / min; injection volume: 20 μL; mobile phase: 0.1% formic acid aqueous solution (A) and methanol (B); detection wavelength 320 nm; gradient elution program: 0-5 min, 10-15% B; 5-25 min, 15-60% B; 25-27 min, 60-10% B (v / v).

[0072] 3. Mass spectrometry parameters: Agilent Poroshell 120SB-Aq (3.0*150mm, 2.7μm) was used. 0.1% formic acid aqueous solution-methanol was used as the mobile phase. The linear elution conditions were 0-5min, 10-15% B; 5-25min, 15-60% B; 25-27min, 60-10% B (v / v). The ESI source conditions were: dry gas (N2) flow rate 8.0L / min; collision energy, 35eV; spray voltage 3.5kV; capillary temperature: 320℃; auxiliary gas heater temperature, 300℃; sheath gas flow rate: 35arb; auxiliary gas flow rate: 15arb; sweep gas flow rate: 5arb; auxiliary gas, sheath gas, and sweep gas were all high-purity nitrogen. All operations and data analysis were performed in positive ion mode.

[0073] 4. Test results:

[0074] The CmC2'H protein catalyzed reaction is described in detail in Figure 2 The catalytic product is umbelliferone with a molecular weight of 162.14. Figure 3 ) It can be seen that CmC2'H protein can catalyze the production of umbelliferone from p-coumaroyl-CoA (the upper figure is the result of enzyme activity test, and the lower figure is the standard product). The results of enzyme catalytic kinetics show (as shown in Table 1) that CmC2'H in Cnidium monnieri has the highest catalytic efficiency.

[0075]

[0076] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to the above embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A hydroxylase, the amino acid sequence of which is shown in SEQ ID NO.

2.

2. A hydroxylase gene, the sequence of which is a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.2, or a sequence that is fully complementary to the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.

2.

3. A recombinant expression vector encoding the hydroxylase according to claim 1.

4. A transgenic recombinant bacterium expressing the hydroxylase according to claim 1.

5. Use of the hydroxylase according to claim 1 in the synthesis of coumarin compounds.

6. Use of the hydroxylase gene according to claim 2 in synthesizing coumarin compounds.

7. Use of the expression vector according to claim 3 in synthesizing coumarin compounds.

8. A method for synthesizing umbelliferone, the method comprising the steps of: 1) obtaining the hydroxylase as claimed in claim 1; 2) Using the hydroxylase in step 1) to catalyze the synthesis of umbelliferone in an enzyme activity reaction system.

9. A method for synthesizing umbelliferone according to claim 8, characterized in that: In step 1), the hydroxylase is obtained by prokaryotic expression or by chemical synthesis.

10. A method for synthesizing coumarin compounds, the method comprising the following steps: 1) obtaining the hydroxylase as claimed in claim 1; 2) using the hydroxylase in step 1) to catalyze the synthesis of umbelliferone in an enzyme activity reaction system; 3) The umbelliferone synthesized in step 2 is further used as a substrate for synthesizing other coumarin compounds.

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