Coumarin o-prenyltransferase and use thereof

Abstract

The application discloses a coumarin O-isopentenyltransferase belonging to a UbiA family and an application thereof, the coumarin O-isopentenyltransferase gene is identified from a Rutaceae plant, and in addition, it is proved through experiments that the coumarin O-isopentenyltransferase gene can be stably expressed in a tobacco and an escherichia coli and the like heterologous system, and can efficiently catalyze the conversion of umbelliferone to generate auraptene; the advantage of the application lies in that, as the first identified O-isopentenyltransferase capable of catalyzing the synthesis of auraptene, the PtPT can be used as a key biological catalysis element for constructing an efficient heterologous synthesis system of auraptene, so that the biological manufacturing efficiency is significantly improved, and the application has good industrialization application potential.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology and involves key enzyme genes involved in the synthesis pathway of coumarin isopentenyl compounds. More specifically, it involves the identification, functional verification and application of coumarin O-isopentenyltransferase PtPT7. Background Technology

[0002] UbiA family membrane-bound aromatic isopentenyltransferases are an important superfamily of membrane proteins. In plants, UbiA-PT can catalyze the transfer of isopentenyl units to the aromatic nucleus to form C-C or CO bonds, participating in the synthesis of important secondary metabolites such as phenols, phenylpropanoids, and flavonoids.

[0003] Coumarins are a class of organic heterocyclic compounds with a catechol structure, produced by the further reaction of the intermediate cinnamic acid during phenylpropanoid metabolism. They are mainly distributed in six families, including Apiaceae, Rutaceae, and Fabaceae. Aurapten, also known as 7-geranyl, is a bioactive substance isolated from Rutaceae plants. Modern pharmacological studies have shown that aurapten is a highly efficient molecule with multiple activities, capable of regulating multiple intracellular signaling pathways such as inflammation, cell proliferation, and apoptosis. Due to its excellent anti-inflammatory and antioxidant activities and good safety profile, it shows broad application prospects in the prevention and treatment of various diseases. Isopentenyltransferases play a crucial role in the coumarin biosynthesis pathway. Existing studies have shown that currently reported coumarin isopentenyltransferases are mainly C-PTs that catalyze the formation of C–C bonds, while O-PTs that can transfer isopentenyl units to the oxygen atom of the aromatic parent nucleus to form C–O bonds are rarely reported. Hesperidin, an O-isopentenylated coumarin derivative, has had its biosynthesis involving the UbiA family O-isopentenyltransferase gene, which has not yet been reported. Given the scarcity of research on O-isopentenyltransferases, the key synthetic gene of this biologically important compound remains unclear. Systematic cloning and identification of coumarin O-isopentenyltransferases with novel functions and catalytic site specificity will not only help to elucidate the molecular mechanism of coumarin isopentenylation but also lay an important foundation for the industrial application of the synthetic biology of hesperidin. Summary of the Invention

[0004] To address the problem that the key isopentenyltransferase gene for the synthesis of hesperidin in existing technologies is still unclear, this invention provides an O-isopentenyltransferase PtPT7 and its encoding gene. This gene can be expressed in heterologous systems such as tobacco and *Escherichia coli*, and its encoded protein can specifically catalyze the transfer of geranyl pyrophosphate (GPP) to the 7-OH group of umbelliferone, forming a C–O bond, thereby achieving the biotransformation of umbelliferone into hesperidin. To achieve the above objective, the technical solution of this invention is as follows:

[0005] This invention provides a coumarin O-isopentenyltransferase, PtPT7, whose gene sequence is shown in SEQ ID NO.1. The PtPT7 coumarin O-isopentenyltransferase gene discovered in this invention was cloned from *Citrus reticulata* (also known as *Citrus trifoliata*, a plant in the Rutaceae family), and is a novel coumarin isopentenyltransferase gene. This enriches the types of coumarin isopentenyltransferases catalyzing CO bond synthesis. Furthermore, hesperidin possesses important pharmacological activities such as anticancer, anti-inflammatory, anti-Helicobacter pylori, anti-endotoxin, and neuroprotective properties. The key enzyme discovered in this study will contribute to the heterologous biosynthesis of this substance, promoting its research and application in the field of synthetic biology.

[0006] As with the same inventive concept as the above-described technical solutions, the present invention also seeks protection for the nucleotide sequence of the gene for coumarin O-isopentenyltransferase PtPT7 and the protein encoded therein, the nucleotide sequence being shown in SEQ ID NO.1, and the amino acid sequence of the protein being shown in SEQ ID NO.2; and for protection of a coumarin isopentenyltransferase-derived protein having one or more amino acids substituted, deleted, or added in the amino acid sequence of SEQ ID NO.2 and having O-isopentenyltransferase activity for producing hesperidin.

[0007] Furthermore, the use of the coumarin O-isopentenyltransferase gene in heterologous expression in other organisms can catalyze the isopentenylation reaction of umbelliferone to produce bioactive hesperidin. Umbelliferone is a key intermediate in coumarin. This invention demonstrates that the recombinant PtPT7 protein has catalytic activity towards umbelliferone. This is the first discovery of a coumarin O-isopentenyltransferase gene that uses umbelliferone as a substrate and the hydroxyl group at position 7 as a binding site to bind geraniol to form hesperidin.

[0008] The present invention, by adopting the above technical solution, has the following beneficial effects:

[0009] This invention cloned the coumarin O-isopentenyltransferase gene from *Citrus trifoliata* (Rutaceae family) and systematically identified its function. It was found that this gene can be overexpressed in *Nicotiana benthamiana* leaves to produce an active recombinant protein that catalyzes the isopentenylation of umbelliferone to generate hesperidin at a content of 1.505 μg / g DW. Simultaneously, this gene can also produce an active recombinant protein in *Escherichia coli*, which, through in vitro protein incubation, catalyzes the isopentenylation of umbelliferone to generate hesperidin. Attached Figure Description

[0010] Figure 1 This is a phylogenetic tree diagram showing the coumarin O-isopentenyltransferase sequence and isopentenyltransferase sequences associated with other plants.

[0011] Figure 2 This is a comparative analysis of the amino acid sequences of coumarin isopentenyltransferase and other plant coumarin isopentenyltransferases. The two conserved motifs of plant coumarin isopentenyltransferase, NQxxDxxxD and KDxxDx (D / E)GD, are represented by wireframes.

[0012] Figure 3 To identify the heterologous expression product of recombinant protein PtPT7 in Nicotiana benthamiana and the MS and MS / MS spectra of the product hesperidin using UHPLC-HRMS / MS technology.

[0013] Figure 4 To identify the in vitro enzyme activity product chromatogram of recombinant protein PtPT7 and the MS and MS / MS chromatograms of the product orange peel oil lactone using UHPLC-HRMS / MS technology.

[0014] Figure 5 To identify the MS / MS spectrum of the heterologous expression product of the MePT1 gene in Nicotiana benthamiana using UHPLC-HRMS / MS technology, it was found that the byproduct had a peak similar to that of hesperidin at [M]+ 229.113, but the MS / MS results showed that it was not hesperidin. Detailed Implementation

[0015] The present invention will be further described below with reference to the accompanying drawings. Unless otherwise specified, the materials, reagents, and instruments used in the following embodiments of the present invention are all conventional materials, reagents, and instruments in the art and are commercially available. Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art.

[0016] This invention discovers a coumarin O-isopentenyltransferase, named PtPT7, whose gene sequence is shown in SEQ ID NO.1, where ATG is the start codon and TGA is the stop codon. The protein encoded by this gene has the amino acid sequence shown in SEQ ID NO.2. Its use is to catalyze the isopentenylation reaction of umbelliferone by heterologous expression in other organisms, producing a bioactive hesperidin.

[0017] This gene was identified and cloned in trifoliate orange (Citrus aurantium), a member of the Rutaceae family. The following describes how this gene was cloned from trifoliate orange and how it was determined that it can catalyze the conversion of umbelliferone into hesperidin.

[0018] I. Materials and Methods

[0019] 1.1 Plant materials and growing environment

[0020] *Citrus trifoliata*, a member of the Rutaceae family, was cultivated in the resource nursery of the National Citrus Breeding Center at Huazhong Agricultural University. Roots from one-year-old *Citrus trifoliata* were harvested, washed with distilled water, and immediately flash-frozen in liquid nitrogen. They were then stored at -80°C for subsequent RNA extraction. *Nicotiana benthamiana*, grown in nutrient soil, was used in experiments after 6 weeks of growth under 16 hours of light, 8 hours of darkness, and 25°C.

[0021] 1.2 Vectors and strains used in the experiment

[0022] Escherichia coli strains DH5α and BL21(DE3) and Agrobacterium tumefaciens strain GV3101 were purchased from Shanghai Weidi Biotechnology Co., Ltd., protein expression vector pETDuet-1 was purchased from Beijing Qingke Biotechnology Co., Ltd., overexpression vector pEAQ-HT (Sainsbury et al., 2009), and RN38-EASYspinPlus plant RNA rapid extraction kit were purchased from Beijing Adley Biotechnology Co., Ltd.

[0023] 1.3 Chemical Standards and Reagents

[0024] Coumarin standards aurapten (CAS: 495-02-3) and umbelliferone (CAS: 93-35-6) were purchased from Shanghai Yuanye Biotechnology Co., Ltd. GPP was purchased from Sigma-Aldrich.

[0025] 1.4 Extraction and Reverse Transcription of Total RNA from Plants

[0026] Fresh trifoliate orange root was used to extract total RNA using the RN38-EASYspin Plus Plant RNA Rapid Extraction Kit. The resulting RNA solution was tested for integrity, concentration, and purity. Reverse transcription experiments were performed using HiScript II Q Select RTSuperMix for qPCR (+gDNA wiper) (Vazyme, Nanjing, China). The reverse transcription product (cDNA reaction solution) was first quality-verified, and PCR amplification was performed using actin gene primers. The qualified cDNA was stored at -20°C for later use.

[0027] 1.5 Design of primers for amplifying the O-isopentenyltransferase PtPT7 gene

[0028] PtPT7 F1:5′-ATGGTTCATATGCATTCATGTT-3′, SEQ ID NO.3;

[0029] PtPT7 R1:5′-TCAACGTACAAATGAATAAGGAG-3′, SEQ ID NO.4;

[0030] The primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0031] 1.6 Amplification of the full-length gene sequence of coumarin O-isopentenyltransferase PtPT7

[0032] Using *Citrus aurantium* root cDNA as a template, PCR amplification was performed using amplification primers. After completion, the PCR product was purified using the EasyPure® Quick Gel Extraction Kit (TransGen Biotech, Beijing, China) and then cloned into the pCE2TA / Blunt-Zero Vector (Vazyme, Nanjing, China). The vector was then transformed into *E. coli* competent cells DH5α, and positive clones were selected for sequencing (Beijing Qingke Biotechnology Co., Ltd.). PtPT7 gene clones with correct sequences were selected and preserved for subsequent expression vector construction.

[0033] The open reading frame (ORF) of the coumarin O-isopentenyltransferase PtPT7 gene obtained by sequencing is 1182 bp in length, and its nucleotide sequence is shown in SEQ ID NO.1; it encodes 393 amino acids, and its amino acid sequence is shown in SEQ ID NO.2.

[0034] 1.7 Construction of overexpression vectors and protein expression vectors

[0035] Using the obtained gene sequence of coumarin O-isopentenyltransferase PtPT7 as a template, adapter primers for the overexpression vector pEAQ-HT and the protein expression vector pETDuet-1 were designed as follows:

[0036] pEAQ-PtPT7 F1:5′-tgcccaaattcgcgaccggtATGGTTCATATGCATTCATGTT-3′, SEQID NO.5;

[0037] pEAQ-PtPT7 R1:5′-ccagagttaaaggcctcgagTCAACGTACAAATGAATAAG-3′, SEQID NO.6;

[0038] pETDuet1-PtPT7 F1:5′-caccatcatcaccacagccagATGGTTCATATGCATTCATGTT-3′,SEQ ID NO.7;

[0039] pETDuet1-PtPT7 R1:5′-caggcgcgccgagctcgaattTCAACGTACAAATGAATAAG-3′,SEQ ID NO.8;

[0040] The PtPT7 gene was directly ligated into the overexpression vector pEAQ-HT (restriction endonuclease sites AgeI and XhoI) and the protein expression vector pETDuet-1 (restriction endonuclease sites BamHI and EcoRI) using a double enzyme digestion method. The resulting clones were transformed into *E. coli* competent cells DH5α, and positive clones were selected for sequencing. After confirming the sequence was correct, pEAQ-PtPT7 was used for genetic transformation of *Agrobacterium tumefaciens* GV3101, and pETDuet1-PtPT7 was used for genetic transformation of *E. coli* BL21(DE3).

[0041] 1.8 Heterologous expression experiment of Tobacco Benedict

[0042] (1) pEAQ-PtPT7 (GV3101) and pEAQ-Ev (GV3101) were added to LB containing kana + rif resistance, respectively, and cultured on a shaker at 28℃ and 200 rpm until OD. 600 : 0.6-0.8;

[0043] (2) Centrifuge the bacterial culture at 8000 rpm, remove the supernatant, and resuspend the precipitate in tobacco infection solution (10 mM MES, 10 mM MgCl2, pH 5.6). OD 600Adjust the pH to 0.6-0.8, add 150 μM As, and incubate at 28°C for 2 hours.

[0044] (3) Inject the tobacco infection solution into the tobacco leaves, inject the standard umbelliferone (200 μM) 2 days later, and continue to culture in the growth chamber for 2 days before collecting the leaves;

[0045] (4) Immediately freeze the tobacco leaves with liquid nitrogen and freeze-dry the samples in a freeze dryer. Store the freeze-dried samples at -80℃.

[0046] 1.9 Extraction of coumarin from tobacco leaves

[0047] (1) Grind the freeze-dried tobacco leaves into a fine powder, and mix 0.1g of the dry powder with 1mL of pure methanol (HPLC) and sonicate in an ultrasonic water bath for 30min.

[0048] (2) Centrifuge at 12000 rpm for 10 min, transfer 700 μL of supernatant to a new 2 mL centrifuge tube, and then resuspend the precipitate with 650 μL of pure methanol (HPLC);

[0049] (3) The second extraction was carried out in an ultrasonic water bath for 30 min, followed by centrifugation at 12000 rpm for 10 min.

[0050] (4) Mix the supernatant from the second extraction with the supernatant from the first extraction and evaporate it to dryness at 30°C for 2 hours in a vacuum concentrator;

[0051] (5) After vacuum concentration, the sample was resuspended in 250 μL of 75:25 methanol (HPLC) / ultrapure water, sonicated in an ultrasonic water bath for 10 min, and centrifuged at 12000 rpm for 10 min to remove the remaining fragments.

[0052] (6) Use a 1mL sterile syringe to draw 200μL, filter it through an organic filter membrane with a pore size of 0.22μm, and put it into a sample vial. Store it at -20℃ until it is ready for testing.

[0053] 1.10 In vitro enzyme activity assay

[0054] (1) pETDuet1-PtPT7 (BL21) and pETDuet1-Ev (BL21) were added to LB containing amp resistance and cultured on a shaker at 37℃ and 200rpm until OD600: 0.6-0.8.

[0055] (2) Add 0.4 mM IPTG to the bacterial culture and incubate on a shaker at 16°C and 120 rpm for 14-16 h;

[0056] (3) Centrifuge the bacterial culture at 8000 rpm to enrich the colonies, and resuspend them in 1×PBS;

[0057] (4) Use an ultrasonic cell disruptor to disrupt the cells (30 min total, 4 s working time, 4 s rest time).

[0058] (5) After the solution was crushed, it was centrifuged at 7000 rpm for 1 h at 4℃, and the supernatant was collected as crude enzyme.

[0059] (6) The components and final concentrations of the PtPT7 enzyme activity reaction are as follows: 100 μL crude enzyme, 200 μM umbelliferone, 200 μM MgCl2, 100 μM GPP, 50 mM Tris-HCl, and pure water is added to bring the total system to 200 μL.

[0060] (7) Place the protein incubation system in a shaker at 25°C and 220 rpm for 5 h;

[0061] (8) After incubation, add an equal volume of pure methanol (HPLC) and shake for 1 min to terminate the reaction, then centrifuge at 12000 rpm for 1 min.

[0062] (9) Use a 1mL sterile syringe to draw 200μL, filter it through an organic filter membrane with a pore size of 0.22μm, and put it into a sample vial. Store it at -20℃ until it is ready for testing.

[0063] 1.11 UHPLC-HRMS / MS detection conditions

[0064] Coumarin samples extracted from the heterologous expression experiment of Tobacco Benzovia and samples from the in vitro enzyme activity experiment were taken in appropriate amounts and analyzed by LC-HRMS.

[0065] The instrument used was an ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS / MS) system (QExactive Plus, Thermo Fisher Scientific, San Jose, CA, USA), and positive and negative ion operation modes were performed.

[0066] Mobile phase: Phase A is a 0.1% formic acid aqueous solution; Phase B is a 0.1% formic acid methanol solution.

[0067] Elution gradient (A:B; v / v): 0-0.5 min, 98:2; 0.5-15 min, 98:2; 15-26 min, 70:30; 26-28 min, 2:98; 28.1-30 min, 98:2, flow rate 0.4 mL / min, injection volume 3 μL.

[0068] The separation column used for sample analysis is a Hypersil GOLD HPLC column (Thermo), with dimensions of 100×2.1 mm and a particle size of 3.0 μm. It needs to operate in a constant temperature environment of 40°C.

[0069] II. Results

[0070] 2.1 PtPT7 heterologous expression in tobacco

[0071] This invention clones the PtPT7 gene into the overexpression vector pEAQ-HT. The plasmid carrying the PtPT7 gene (pEAQ-PtPT7) is transformed into Agrobacterium tumefaciens GV3101, and its straw infection solution is injected into tobacco leaves. The mixture is then incubated with the reaction substrate to conduct a heterologous expression experiment in tobacco. Coumarin is extracted from the tobacco leaves, and the substrate and product are detected using HPLC and LC-HRMS. The results show that tobacco injected with pEAQ-PtPT7 and umbelliferone produced hesperidin, while tobacco injected with pEAQ-Ev and umbelliferone did not produce hesperidin, indicating that PtPT7 has a catalytic function for the 7-position hydroxyl oxygen of umbelliferone. Figure 3 The peak area of ​​the product was substituted into the standard curve of hesperidin for calculation, and the result showed that the product content was 1.505 μg / g DW. 2.2 In vitro enzyme activity verification of PtPT7

[0072] This invention clones the PtPT7 gene into the protein expression vector pETDuet-1. The plasmid carrying the PtPT7 gene (pETDuet1-PtPT7) was transformed into *E. coli* BL21(DE3), and IPTG-induced protein was added. The crude enzyme was extracted and co-incubated with substrate and cofactor for in vitro enzyme activity experiments. The incubated system was terminated and extracted with methanol, and the substrate and product were detected using HPLC and LC-HRMS. The results showed that hesperidin was produced in the system containing pETDuet1-PtPT7 crude protein and umbelliferone substrate, while no hesperidin was produced in the system containing pETDuet1-Ev crude protein and umbelliferone. This further demonstrates that PtPT7 has a catalytic function for the 7-position hydroxyl group of umbelliferone. Figure 4 ).

[0073] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A coumarin O-isopentenyltransferase, characterized in that, The coumarin O-isopentenyltransferase is named PtPT7, and its amino acid sequence is shown in SEQ ID NO.

2.

2. The coumarin isopentenyl transferase according to claim 1, characterized in that, The nucleotide sequence of the O-coumarin isopentenyltransferase gene is shown in SEQ ID NO.

1.

3. An O-coumarin isopentenyltransferase-derived protein, characterized in that, It is a coumarin isopentenyltransferase-derived protein that has one or more amino acids substituted, deleted, or added in the amino acid sequence of SEQ ID NO.2 and has O-coumarin isopentenyltransferase activity.

4. The application of the O-coumarin isopentenyltransferase according to claim 1 in the synthesis of orange peel oil lactone.

5. The application according to claim 1, characterized in that, The O-coumarin isopentenyltransferase catalyzes the transfer of geranylpyrophosphate (GPP) to the 7-hydroxyl group (7-OH) of umbelliferone, resulting in isopentenylation and the formation of orange peel oil lactone.

6. An overexpression vector pEAQ-PtPT7, characterized in that, The gene containing the O-coumarin isopentenyltransferase PtPT7 as described in claim 1 and the pEAQ-HT vector / other plant overexpression vector.

7. A protein expression vector pET-Duet1-PtPT7, characterized in that, The gene containing the O-coumarin isopentenyltransferase PtPT7 as described in claim 1 and the pET-Duet1 vector / other prokaryotic expression vector.

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