The 4-coumaric acid-coenzyme A ligase gene Gl4CL of *Hypericum perforatum* and its encoded product and applications.

Abstract

This invention belongs to the field of genetic engineering, and specifically relates to a 4-coumaric acid-coenzyme A ligase gene Gl4CL from *Gynostemma pentaphyllum*, its encoded product, and its applications. This invention is the first to clone and prepare the 4-coumaric acid-coenzyme A ligase gene Gl4CL from the medicinal plant *Gynostemma pentaphyllum*. The nucleotide sequence of this gene is shown in SEQ ID NO.1, and the amino acid sequence of the product encoded by this nucleotide sequence is shown in SEQ ID NO.2. The 4-coumaric acid-coenzyme A ligase gene Gl4CL is a key regulatory gene in the flavonoid biosynthesis pathway of *Gynostemma pentaphyllum*. This enzyme can be used in the preparation of p-coumaryl-CoA using p-coumaric acid as a substrate, further promoting the biosynthesis of flavonoid compounds. This invention can provide an effective method to meet the huge market demand for flavonoid compounds.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, and specifically relates to the gene Gl4CL of 4-coumaric acid coenzyme A ligase in *Hypericum perforatum*, its encoded product, and its applications. Background Technology

[0002] *Glechoma longituba* (Nakai) Kupr., a perennial herb belonging to the genus *Glechoma* in the family Lamiaceae, is used medicinally for its dried aerial parts, known as *Lianqiancao*. *Glechoma longituba* is mainly distributed in damp, shady places such as forest edges and streamsides, and has the effects of promoting diuresis and relieving strangury, clearing heat and detoxifying, and dispersing blood stasis and reducing swelling. *Lianqiancao* has various pharmacological effects, including diuresis, choleretic effects, lipid-lowering and litholytic effects, and antibacterial and anti-inflammatory properties, and is often used to treat various diseases such as cholelithiasis, urolithiasis, and urinary tract infections.

[0003] The chemical composition of *Gynostemma pentaphyllum* is diverse, including volatile oils, flavonoids, terpenes, and phenolic acids. Flavonoids are the main active components in *Gynostemma pentaphyllum*, and 4-coumarate-CoA ligase (4CL) is a key enzyme in the flavonoid biosynthesis pathway. It catalyzes the formation of corresponding coenzyme A esters from cinnamic acid, coumaric acid, caffeic acid, and ferulic acid, thereby directing metabolism towards different branches such as flavonoids, isoflavones, flavanones, and anthocyanins. However, the gene for 4-coumarate-CoA ligase (Gl4CL) in the flavonoid biosynthesis pathway of *Gynostemma pentaphyllum* has not yet been isolated and identified. Summary of the Invention

[0004] To address the above-mentioned problems, one of the objectives of this invention is to propose a 4-coumaric acid coenzyme A ligase gene Gl4CL from *Hypericum perforatum*, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0005] The second objective of this invention is to propose a product encoded by the 4-coumaric acid coenzyme A ligase gene Gl4CL of the aforementioned *Hypericum perforatum*, wherein the product comprises amino acids, polypeptides, or proteins.

[0006] Furthermore, the amino acid sequence of the product is shown in SEQ ID NO.2.

[0007] A third objective of this invention is to provide a recombinant expression vector containing the *Hypericum pertussis* 4-coumaric acid-coenzyme A ligase gene Gl4CL. Further, the target gene in the recombinant expression vector is amplified by PCR using the cDNA of the *Hypericum pertussis* 4-coumaric acid-coenzyme A ligase gene Gl4CL as a template and SEQ ID No. 5 and SEQ ID No. 6 as primers. The *Hypericum pertussis* 4-coumaric acid-coenzyme A ligase gene Gl4CL sequence is inserted into the BamHI single restriction site of the pET-30a expression vector, and the vector is ligated and transformed into *E. coli* to obtain the recombinant expression vector.

[0008] The fourth objective of this invention is to provide a specific primer pair for constructing the recombinant expression vector described above; further, the nucleotide sequence of the upstream primer of the specific primer pair is shown in SEQ ID NO.5, and the nucleotide sequence of the downstream primer of the specific primer pair is shown in SEQ ID NO.6.

[0009] The fifth objective of this invention is to provide a recombinant engineered bacterium containing the *Hypericum perforatum* 4-coumaric acid coenzyme A ligase gene Gl4CL or the recombinant expression vector described above.

[0010] The sixth objective of this invention is to provide a host cell containing the 4-coumaric acid coenzyme A ligase gene Gl4CL from *Hypericum perforatum* or containing the recombinant expression vector described above.

[0011] Furthermore, the host cells include BL21(DE3) cells.

[0012] The seventh objective of this invention is to propose the application of the aforementioned 4-coumaric acid coenzyme A ligase gene Gl4CL from *Hypericum perforatum*, the aforementioned recombinant expression vector, the aforementioned recombinant engineered bacteria, or the aforementioned host cell in the preparation of flavonoid compounds.

[0013] Furthermore, the application is to synthesize p-coumaryl-CoA, a precursor in the flavonoid synthesis pathway, using p-coumaric acid as a substrate; specifically, the p-coumaryl-CoA gene Gl4CL of *Huoxuedan* can be transferred into cells and the p-coumaryl-CoA of *Huoxuedan* can be used to promote the biosynthesis of flavonoid compounds.

[0014] The beneficial effects of this invention are:

[0015] This invention is the first to clone and prepare the 4-coumaric acid-co-A ligase gene Gl4CL from the medicinal plant *Gynostemma pentaphyllum*. The coumaroyl-CoA gene Gl4CL is a key regulatory gene in the biosynthetic pathway of flavonoids in *Gynostemma pentaphyllum*, and can be applied to prepare coumaroyl-CoA using coumaric acid as a substrate, and further prepare flavonoid components. This can provide an effective method to meet the huge market demand for flavonoid compounds.

[0016] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. The objects and other advantages of the invention may be realized and obtained by means of the structures pointed out in the description, claims and drawings. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 The image shown is an agarose gel electrophoresis diagram of the effect of Huoxuedan on the coumaroyl-CoA gene Gl4CL in an embodiment of the present invention.

[0019] Figure 2 This invention illustrates the predictive analysis of the functional domain of coumaroyl-CoA Gl4CL by Huoxuedan in an embodiment of the present invention.

[0020] Figure 3 This invention illustrates the predictive analysis of the transmembrane domain of coumaroyl-CoA Gl4CL by Huoxuedan in an embodiment of the present invention.

[0021] Figure 4 This invention illustrates the predictive analysis of the secondary structure of coumaroyl-CoA Gl4CL by Huoxuedan in an embodiment of the present invention;

[0022] Figure 5 The phylogenetic tree of coumaroyl coenzyme A Gl4CL in the embodiment of the present invention is shown;

[0023] Figure 6 This invention illustrates the predictive analysis of the tertiary structure of coumaroyl-CoA Gl4CL by Huoxuedan in an embodiment of the present invention;

[0024] Figure 7 The image shown is a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) image of coumaroyl coenzyme A Gl4CL protein by Huoxuedan in an embodiment of the present invention.

[0025] Figure 8 The chromatograms of the pET-30a empty carrier and the reaction of p-coumaric acid catalyzed by coumaroyl coenzyme AGL4CL in the embodiments of the present invention are shown. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] The techniques used in these embodiments are conventional methods well-known to those skilled in the art, and the raw materials used are all commercially available products. Some of the raw materials are as follows:

[0028] Rapid high-fidelity DNA polymerase FastPfu Fly DNA Polymerase, Quick Gel Extraction Kit, HiPure Plasmid MiniPrep Kit, Seamless Splicing Kit Seamless Cloning and Assembly Kit, Restriction Endonuclease BamHI, protein purification Ni-NTA Resin, cloned competent cells Trans1-T1, expression-competent cells BL21(DE3), protein molecular weight standard Blue II Protein Marker (14-120kDa) was purchased from Beijing TransGen Biotechnology Co., Ltd.

[0029] DNA Marker molecular weight standards (100-2000bp), isopropyl-β-D-thiogalactoside (IPTG), and SDS-PAGE denaturing acrylamide gel rapid preparation kit were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0030] Anhydrous magnesium chloride was purchased from Shanghai Mairui Biochemical Technology Co., Ltd.

[0031] 5-Adenosine triphosphate disodium salt hydrate (ATP) and coenzyme A hydrate were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0032] The standard products p-coumaric acid and p-coumaroy l-CoA were purchased from Sichuan Jingcui Tiancheng Pharmaceutical Technology Co., Ltd.

[0033] Example 1

[0034] Cloning of the 4-coumaric acid coenzyme A ligase gene Gl4CL from Huoxuedan (a traditional Chinese medicine):

[0035] Primers were designed based on the Gl4CL sequence from the transcriptome of *Hedyotis diffusa*, and *Hedyotis diffusa* cDNA was used as a template for PCR amplification. The primer sequences are shown in Table 1.

[0036] Table 1

[0037] Primer name Serial Number Base sequence (5'→3') upstream primer SEQ ID NO.3 ATGTTGTCGGTGGCCTCCGCCGAAG Downstream primer SEQ ID NO.4 TTAAGAGGTGGAGGAAGCTGCAAGT

[0038] The amplification system (50 μL) is as follows: rapid high-fidelity DNA polymerase FastPfu Fly DNAPolymerase 1μL, 25 μL FastPfu Fly Reaction Mix, 1 μL template cDNA, 1 μL each of upstream and downstream primers, and 21 μL sterile water.

[0039] Reaction conditions: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 50 s, 55±5℃ annealing for 1 min, 72℃ extension for 1 min, 30 cycles followed by 72℃ extension for 10 min, and storage at 4℃.

[0040] The clone of coumaroyl-CoA gene Gl4CL by Huoxuedan was obtained according to the above amplification system and conditions. The agarose gel electrophoresis result of Huoxuedan on coumaroyl-CoA gene Gl4CL is shown below. Figure 1 As shown, Figure 1 The "M" in the diagram represents the marker (the molecular weight standard for DNA markers is 100-2000 bp). The target gene Gl4CL fragment size was approximately 1700 bp, which was expected. The target band was recovered using the EasyPure Quick Gel Extraction Kit. The amplified product was ligated into the cloning vector pEASY-Blunt Zero and transformed into *E. coli* Trans1-T1 competent cells. Single colonies were selected, expanded, and validated by colony-linked PCR. Positive colonies were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0041] Bioinformatics analysis of the 4-coumaric acid coenzyme A ligase gene Gl4CL from *Hypericum perforatum*:

[0042] The open reading frame (ORF) of the coumaroyl-CoA Gl4CL gene obtained in this invention is 1719 bp in length, and its sequence is shown in SEQ ID No. 1. The ORF of the Gl4CL gene encodes 572 amino acids, as shown in SEQ ID No. 2.

[0043] The Gl4CL gene sequence was subjected to nucleotide homology searches in the Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDStranslation+PDB+Swissprot+Superdate+PIR databases using the BLAST program in the NCBI database. This gene showed high homology at the amino acid level to 4CL from other species.

[0044] The Gl4CL protein contains an active site, an AMP binding site, and a coenzyme A binding site, and possesses the conserved PLN02246 domain. It belongs to the 4CL gene family, such as... Figure 2 As shown. Gl4CL lacks a transmembrane domain and is an extramembrane protein, such as... Figure 3 As shown. The secondary structure of coumaroyl-CoA Gl4CL in Huoxuedan consists of an α-helix, an extended chain, and random coils, as shown. Figure 4 As shown. A Neighbor-joining phylogenetic tree was constructed using MEGA 6.0 software with the neighbor-joining method based on the Gl4CL amino acid sequences, as shown. Figure 5 As shown, the amino acid sequence of coumaroyl-CoA ligase AG14CL from *Polygonum cuspidatum* is most closely related to that of 4-coumarate:CoA ligase from *Prunella vulgaris* (Lamiaceae family). Homology modeling of AG14CL was performed using the crystal structure of *Populus tomentosa* 4CL protein (PDBID: 3a9u.1.A) as a model, and the AG14CL protein showed 62.57% identity with the template protein. Figure 6 The monomer structure of coumaroyl-CoA Gl4CL protein of Huoxuedan is obtained.

[0045] Example 2

[0046] Construction of a recombinant expression vector system for the 4-coumaric acid coenzyme A ligase gene Gl4CL from *Hypericum perforatum*:

[0047] Using the cDNA of the Gl4CL gene as a template, restriction endonucleases were selected. Using BamHI (hereinafter referred to as BamHI enzyme) as the restriction site, upstream and downstream primers with restriction sites were designed (as shown in Table 1) for PCR amplification. The underlined parts in the primers represent the base sequences on the vector.

[0048] Table 2. Base sequences of specific upstream and downstream primers.

[0049] Primer name sequence name Base sequence (5'-3') Gl4CL_BamHI_F SEQ ID NO.5 AGGCCATGGCTGATATCGGAATGTTGTCGGTGGCCTCCGCCGAAG Gl4CL_BamHI_R SEQ ID NO.6 CGACGGAGCTCGAATTCGGATTAAGAGGTGGAGGAAGCTGCAAGT

[0050] The *Gl4CL* gene ligase for 4-coumarate-coenzyme A ligase from *Heliotropium indicum* and the pET30a vector were digested with BamHI and subjected to agarose gel electrophoresis. The digested bands were recovered using a Quick Gel Extraction Kit. The recovered target fragment was then seamlessly spliced ​​with the prokaryotic expression vector pET30a using a seamless splicing kit. The Seamless Cloning and Assembly Kit was used for ligation at 50°C for 30 min. The ligation product was transformed into *E. coli* clonal competent cells Trans1-T1, and single colonies were picked for colony PCR detection. Positive bacterial cultures with correct sequencing were expanded and processed using a plasmid extraction kit. Extraction was performed using the Seamless Cloning and Assembly Kit to obtain the constructed recombinant expression vector pET30a-Gl4CL.

[0051] Example 3

[0052] Induction and protein purification of genetically engineered bacteria:

[0053] The recombinant expression vector pET30a-Gl4CL was transformed into competent BL21(DE3) cells. Transformed cells were plated on LB solid medium containing 50 mg / L kanamycin to screen for positive clones. Single positive colonies were selected and inoculated into LB liquid medium containing 50 mg / L kanamycin and cultured overnight. The culture was then scaled up at a 1:100 ratio until the bacterial culture reached OD500. 600 When the concentration reached between 0.4 and 0.6, IPTG (isopropyl-β-D-thiogalactopyranoside) was added to a final concentration of 0.8 mM, and the mixture was slowly induced for 12 h in a shaker at 16 °C. The pET30a empty vector was treated in the same manner as a blank control.

[0054] The induced bacterial culture was centrifuged at 4°C and 5000×g for 10 min, the supernatant was discarded, and the cells were washed three times with 5 mL PBS. After washing, the cells were centrifuged at 5000×g for 10 min, the supernatant was discarded, and the cells were immediately cooled on ice. The cells were resuspended in 5 mL PBS and sonicated on ice. After sonication, the cells were centrifuged at 4°C and 10000×g for 15 min, and 50 μL of the supernatant was analyzed by 10% SDS-PAGE electrophoresis (the SDS-PAGE gel used for analysis was prepared using a rapid preparation kit for denaturing acrylamide gels). The results are as follows: Figure 7 As shown, the recombinant expression vector pET30a-Gl4CL, after induction, expressed a soluble protein with a band located between 75 kDa and 100 kDa, consistent with the molecular weight of the Gl4CL protein (using Blue). II Protein Marker (14-120kDa) is used as a protein molecular weight standard.

[0055] Aspirate 1 mL of Gl4CL protein supernatant and mix it with 500 μL of washed protein for purification. The Ni-NTAResin mixture was incubated with shaking in an ice bath for 2 hours. After incubation, the mixture was centrifuged at 4°C and 500×g for 5 minutes, the supernatant was discarded, and 1 mL of His Buffer was added to wash away impurities. This process was repeated three times. A gradient elution of different concentrations of imidazole buffer (20 mM, 100 mM, 300 mM, 500 mM) was then performed, adding 200 μL each time. The mixture was centrifuged at 4°C and 500×g for 5 minutes, and the supernatant was collected to obtain purified Gl4CL protein. The Gl4CL protein was analyzed by 10% SDS-PAGE electrophoresis, and the results are shown below. Figure 7 As shown, M represents Protein Marker, lane 1 contains the pET-30a empty vector, lane 2 contains whole bacterial culture containing unpurified Gl4CL protein after induction, lane 3 contains the supernatant of purified Gl4CL protein after induction, and lanes 4-7 contain purified Gl4CL protein eluted with 20mM, 100mM, 300mM, and 500mM imidazole solutions, respectively (arrows indicate the target protein). It can be seen that Gl4CL protein can be efficiently eluted with 100mM, 300mM, and 500mM imidazole solutions, and the 300mM imidazole solution yields the largest amount of purified Gl4CL protein with the best purification effect.

[0056] Example 4

[0057] In vitro enzyme function verification:

[0058] 1. Using p-coumaric acid as a substrate and pET-30a empty vector protein as a negative control, the purified GL4CL protein was functionally identified in vitro. The 500 μL enzymatic reaction system included 115 μL 1M Tris-HCl buffer (pH = 7.5), 150 μL 17 mM MgCl2, 125 μL 20 mM ATP, 5 μL 30 mM CoA, 5 μL 20 mM p-coumaric acid, and 100 μL enzyme solution (GL4CL purified protein supernatant). The reaction was terminated by incubating at 30°C for 1 h and then at 100°C for 10 min. After centrifugation at 12000 × g for 10 min, the supernatant was filtered through a 0.22 μm microporous membrane and the product formation was detected by UPLC.

[0059] 2. Using p-Coumaroyl A as a standard, the formation of the enzymatic product was detected by UPLC. UPLC analysis was performed using an Agilent 1290 ultra-high performance liquid chromatograph with an ACQUITY UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm). Gradient elution was performed using 0.1% phosphoric acid (phase A) and acetonitrile (phase B) as the mobile phase. The elution program was: 0–5 min, 5%–10% B; 5–10 min, 10%–15% B; 10–15 min, 15%–25% B; 15–18 min, 25%–75% B; 18–21 min, 75%–95% B; 21–27 min, 95% B. The flow rate was 0.2 mL / min, the detection wavelength was 333 nm, the column temperature was 30 °C, and the injection volume was 5 μL.

[0060] 3. For example Figure 8 As shown, compared with the empty pET30a vector protein, the purified Gl4CL protein catalyzed a new chromatographic peak for the p-coumaric acid substrate at 13.512 min, which coincided with the peak time of the standard p-coumaryl-CoA (13.719 min). Therefore, it can be concluded that the protein expressed and purified by the pET30a-Gl4CL recombinant expression vector has the function of 4-coumaric acid-CoA ligase.

[0061] In summary, this invention clones the gene encoding coumarin-CoA ligase (Gl4CL) from *Hypericum perforatum*, and then uses genetic engineering technology to ligate the Gl4CL gene into a prokaryotic expression vector. Large-scale expression of the Gl4CL protein in host cells can promote the synthesis of coumarin-CoA, an important precursor in the flavonoid synthesis pathway. Using the gene and technology provided by this invention, large-scale synthesis of coumarin-CoA can be achieved in vitro, and the content of flavonoid compounds such as naringenin in *Hypericum perforatum* can also be increased through genetic engineering.

[0062] The SEQ ID NO.1 and SEQ ID NO.2 mentioned in Example 1 are as follows:

[0063] SEQ ID NO.1:

[0064]

[0065] SEQ ID NO.2:

[0066] MLSVASAEAQNPELSSHALQPQPQPQTQSCEQTDHIFVSKLPSIPISNHLPLHTYCFENFSQYPDRPCLLVGSDGKSYSFAETHLLCRRVAAGLSNLGIRKGDVVMALLQNCAEFVFTFMGASMIGAVITTANPFCTSKEIFK QFHASKSKMIVTQSMYVDKLRDTGDDSLVLGEDFYVVTIDAAPEKCLHFSALSEADESAAPDVEISPDDAVALPFSSGTTGLPKGVILTHKSLITSIAQQVDGENPNLYLKADDVVLCVLPLFHIYSLNSVLLCSLRAGAGVL LMQKFEIGALLELIQLHRVSVAAVVPPLVLALAKNPLVDNFDLSSIRMVLSGAAPLGKELEAALLSRLPQAVFGQGYGMTEAGPVLSMSPSFAKVALPTKSGSCGNVVRNAELKVVDPETGCSLPRNQPGEICIRGPQIMKGY LNDAEATARTVDVDGWLHTGDIGYVDEDDDVFIVDRVKELIKFKGFQVPPAELEALLISHSQISDAAVVPQKDEAAGEVPVAFVVPANGSELTEEAVKEFISKQVVFYKRLHKVYFVHAIPKSPSGKILRKDLRAKLAASSTS

[0067] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A 4-coumaric acid-coenzyme A ligase gene Gl4CL from *Hypericum perforatum*, characterized in that... The nucleotide sequence of the gene Gl4CL is shown in SEQ ID NO.

1.

2. A product encoded by the 4-coumaric acid-coenzyme A ligase gene Gl4CL of the *Hypericum perforatum* as described in claim 1, characterized in that, The product includes amino acids, polypeptides, or proteins.

3. The product encoded by the 4-coumaric acid-coenzyme A ligase gene Gl4CL of *Hypericum perforatum* according to claim 2, characterized in that, The amino acid sequence of the product is shown in SEQ ID NO.

2.

4. A recombinant expression vector, characterized in that, The expression vector contains the 4-coumaric acid coenzyme A ligase gene Gl4CL from the blood-activating herb described in claim 1, and is pET-30a.

5. A recombinant engineered bacterium, characterized in that, It contains the 4-coumaric acid coenzyme A ligase gene Gl4CL of *Hypericum perforatum* as described in claim 1, or the recombinant expression vector as described in claim 4.

6. A host cell, characterized in that, It contains the 4-coumaric acid coenzyme A ligase gene Gl4CL of the blood-activating herb as described in claim 1 or the recombinant expression vector as described in claim 4.

7. The host cell according to claim 6, characterized in that, The host cells include BL21 (DE3) cells.

8. The application of the 4-coumaric acid coenzyme A ligase gene Gl4CL of *Hypericum perforatum* as described in claim 1, the recombinant expression vector as described in claim 4, the recombinant engineered bacteria as described in claim 5, or the host cell as described in claim 6 in the preparation of p-coumaric acid coenzyme A, a precursor in the flavonoid synthesis pathway, wherein the application is to synthesize p-coumaric acid coenzyme A, a precursor in the flavonoid synthesis pathway, using p-coumaric acid as a substrate.

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