Acyltransferase csht, coding gene and application thereof

Abstract

The application relates to the field of genetic engineering, discloses an acyltransferase CsHCT, the amino acid sequence of the acyltransferase CsHCT is shown as SEQ ID No: 1; further comprises a coding gene of the acyltransferase CsHCT; and application of the acyltransferase CsHCT and the coding gene thereof to in-vitro synthesis of kaempferol-3-O-(6'-coumaroyl)-glucoside. The application realizes in-vitro enzyme catalytic synthesis of kaempferol-3-O-(6'-coumaroyl)-glucoside, and simultaneously discloses a gene editing method, so that an enzyme capable of catalyzing synthesis of kaempferol-3-O-(6'-coumaroyl)-glucoside is obtained, and the enzyme exhibits certain application prospect in the field of biosynthesis of acylated kaempferol-3-O-glucoside.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, specifically to an acyltransferase CsHCT, its encoding gene, and its applications. Background Technology

[0002] CYP3A4 is a drug-metabolizing enzyme. Kaempferol-3-O-(6'-coumaryl)-glucoside exhibits a significant inhibitory effect on it. The inhibition of its activity by kaempferol-3-O-(6'-coumaryl)-glucoside is of great significance in the study of drug metabolism and drug interactions.

[0003] However, in existing technologies, the extraction of kaempferol-3-O-(6'-coumaryl)-glucoside from samples is generally carried out by separation and purification. This method is complex and the process is cumbersome, resulting in low yield and limited research on it. It is difficult to fully study and utilize this substance. At the same time, due to the complex structure of kaempferol-3-O-(6'-coumaryl)-glucoside, there is currently no simple method for synthesizing kaempferol-3-O-(6'-coumaryl)-glucoside.

[0004] Therefore, there is an urgent need for new technologies to provide new ideas for the in vitro synthesis of kaempferol-3-O-(6'-coumaryl)-glucoside. Summary of the Invention

[0005] The purpose of this invention is to provide an acyltransferase CsHCT, its encoding gene, and its application, in order to solve the technical problem that kaempferol-3-O-(6'-coumaryl)-glucoside is difficult to synthesize in vitro in the prior art.

[0006] To solve the above-mentioned technical problems, the present invention specifically provides the following technical solution:

[0007] The present invention provides an acyltransferase CsHCT, the amino acid sequence of which is shown in SEQ ID No: 1.

[0008] This invention provides a gene encoding the acyltransferase CsHCT.

[0009] This invention provides a primer pair for amplifying the encoding gene of acyltransferase CsHCT, wherein the upstream primer of the primer pair is shown in SEQ ID NO: 2;

[0010] The downstream primer of the nucleotide sequence of the primer pair is shown in SEQ ID NO: 3.

[0011] This invention provides an application of the acyltransferase CsHCT, which catalyzes the in vitro synthesis of kaempferol-3-O-(6'-coumaryl)-glucoside.

[0012] As a preferred embodiment of the present invention, kaempferol-3-O-(6'-coumaryl)-glucoside is prepared in vitro by using the acyltransferase CsHCT to catalyze the in vitro synthesis of kaempferol-3-O-glucoside from coumaroyl coenzyme A and kaempferol-3-O-glucoside.

[0013] This invention provides a method for the in vitro synthesis of kaempferol-3-O-(6'-coumaryl)-glucoside, comprising the following steps:

[0014] Using p-coumaryl coenzyme A as the acyl donor and kaempferol-3-O-glucoside as the acyl acceptor, acyltransferase CsHCT was used as the catalytic enzyme to carry out an enzymatic reaction to obtain kaempferol-3-O-(6'-coumaryl)-glucoside.

[0015] The amino acid sequence of the acyltransferase CsHCT is shown in SEQ ID No: 1.

[0016] This invention provides an application of the encoding gene of acyltransferase CsHCT, through which the in vitro synthesis of kaempferol-3-O-(6'-coumaryl)-glucoside is achieved by prokaryotic expression of the encoding gene of acyltransferase CsHCT.

[0017] This invention provides a method for the in vitro synthesis of acyltransferase CsHCT, comprising the following steps:

[0018] Using the correctly sequenced CsHCT-peasy-Blunt-Zero vector as a template for cloning, the recovered product was ligated into the PMAL-C5X vector that had been digested with restriction endonucleases NdeI and BamHI, and transformed into E. coli DH5α by heat shock to obtain the CsHCT-PMAL recombinant plasmid.

[0019] The CsHCT-PMAL recombinant plasmid was transformed into Escherichia coli BL21(DE3) using a chemical transformation method, and the bacterial cells were disrupted using an ultrasonic disruptor. The disruption time was 10 min, power was 30 W, sonication time was 2 s, pause was 5 s, and after disruption, the cells were centrifuged at 5000 r / min, 4℃ for 15 min using a low-temperature refrigerated centrifuge. The supernatant was collected and purified to obtain the acyltransferase CsHCT.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] This invention provides an acyltransferase CsHCT, which has the function of catalyzing flavonol glycosides. Its amino acid sequence is shown in SEQ ID No: 1. It can efficiently synthesize kaempferol-3-O-(6'-coumaryl)-glucoside via in vitro enzymatic method.

[0022] This invention establishes an in vitro enzymatic reaction system for kaempferol-3-O-(6'-coumaryl)-glucoside based on the acyltransferase CsHCT. Under in vitro conditions, using p-coumaryl coenzyme A as an acyl donor, it catalyzes the synthesis of kaempferol-3-O-glucoside from kaempferol-3-O-glucoside. The product can be detected by high-performance liquid chromatography (HPLC) and identified by liquid chromatography-tandem mass spectrometry (LC-MS / MS), providing a framework for the efficient in vitro enzymatic synthesis of kaempferol-3-O-(6'-coumaryl)-glucoside.

[0023] This invention provides a gene encoding the acyltransferase CsHCT, which is expressed and purified via prokaryotic expression to obtain recombinant CsHCT protein, thereby further disclosing the application of this encoding gene in the in vitro synthesis of kaempferol-3-O-(6'-coumaryl)-glucoside. Attached Figure Description

[0024] To more clearly illustrate the embodiments of the present invention or the technical solutions in 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 merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0025] Figure 1 This invention provides a gel image of the purified target protein shown in Example 2;

[0026] Figure 2 The in vitro enzyme activity chemical spectrum shown in Example 3 is provided for the present invention;

[0027] Figure 3 This invention provides a histogram of the transcriptional level of CsHCT in the experimental group overexpressing CsHCT as shown in Example 4;

[0028] Figure 4 A bar chart showing the content of kaempferol-3-O-(6'-coumaryl)-glucoside in the experimental group overexpressing CsHCT, as shown in Example 4 of this invention, is provided for the present invention. Detailed Implementation

[0029] 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, and 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.

[0030] Example 1: Gene Editing

[0031] Fresh tea leaves were ground into powder under liquid nitrogen. Total RNA was extracted from the tea leaf tissue using a plant RNA extraction kit, and DNAase was used to remove DNA interference. The obtained RNA sample was used as a template for reverse transcription to synthesize the first strand of cDNA. PCR was performed using KOD plus enzyme. The upstream primer for amplifying the encoding gene of the acyltransferase CsHCT is shown in SEQ ID NO: 2, and the downstream primer is shown in SEQ ID NO: 3.

[0032] SEQ ID NO: 2

[0033] ATGAGGATAGAAGTGAGGGAGTC

[0034] SEQ ID NO: 3

[0035] TTATATGTCAAACAAGAACTTCTCA

[0036] The target gene band was obtained by gel electrophoresis to verify its location. The target band was then recovered from the gel and ligated into the cloning vector pEASY-Blunt-Zero, which was then sent to Shanghai Bioengineering Co., Ltd. for sequencing.

[0037] This gene fragment encodes the acyltransferase CsHCT.

[0038] This gene segment has been reported in previous studies as being involved in lignin synthesis, catalyzing the synthesis of substrates such as QA / SA. This invention provides the application of this gene in the in vivo and in vitro synthesis of kaempferol-3-O-(6'-coumaryl)-glucoside. Specifically, this encoding gene is expressed and purified via prokaryotic expression to obtain the CsHCT recombinant protein, which is the catalytic enzyme for the synthesis of kaempferol-3-O-(6'-coumaryl)-glucoside.

[0039] Example 2: Prokaryotic expression and protein purification of CsHCT

[0040] Recombinant vector construction

[0041] The correctly sequenced CsHCT-peasy-Blunt-Zero vector was used as a template for cloning, with primers shown in SEQ ID No: 4 and SEQ ID No: 5. The recovered product was ligated into the PMAL-C5X vector, which had been digested with restriction endonucleases NdeI and BamHI. The vector was then transformed into Escherichia coli DH5α by heat shock. The bacterial culture was plated on LB solid medium containing 100 mg / L ampicillin (Amp) and incubated upside down at 37°C for 12–14 hours. After bacterial sampling, colony PCR, and verification, the CsHCT-PMAL recombinant plasmid was finally obtained.

[0042] SEQ ID No: 4:

[0043] gagggaaggatttcacatatgATGAGGATAGAAGTGAGGGAGTCG

[0044] SEQ ID No: 5:

[0045] acctgcagggaattcggatccTTATATGTCAAACAAGAACTTCTCAAAGA

[0046] Recombinant protein expression:

[0047] 1. Using chemical transformation, the CsHCT-PMAL recombinant plasmid and the PMAL-C5X empty vector plasmid were transformed into E. coli BL21(DE3), plated on solid LB agar containing Ampr resistance, and incubated upside down at 37°C for about 10 hours. Then, single clones of the strain were picked from the solid plates, streaked, and colony PCR was performed for verification. A portion of the bacterial culture from the sample with the correct colony PCR band was selected, and 500 μL of liquid medium containing Ampr was added. The culture was incubated overnight at 37°C with a shaker at 200 rpm. Then, 500 μL of 50% sterile glycerol was added, and the culture was stored at -80°C for later use.

[0048] 2. Select a verified positive monoclonal strain and incubate it in 5 mL of LB liquid medium containing Amp resistance for 10-12 h at 37℃ using a constant-temperature shaker at 200-220 rpm. Take 200 μL of the cultured bacterial solution and dilute it 1:100 into 200 mL of LB liquid medium containing the same resistance for expansion culture. Incubate at 37℃ using a constant-temperature shaker at 200-220 rpm until the OD600 of the bacterial solution reaches 0.6-0.8.

[0049] 3. Take 2 mL of bacterial culture for later use. Add IPTG as an inducer to the remaining culture medium, set the constant temperature shaker to 16℃, and induce for 24 h at 200-220 rpm. Take 2 mL of the induced bacterial culture. Use a refrigerated centrifuge at 4000 g, 4℃, and 15 min to remove the supernatant. Collect the induced bacterial cells, add 20-25 mL of Buffer 1 to the pellet to resuspend the cells, and collect the resuspension in a 50 mL centrifuge tube.

[0050] The specific composition of Buffer 1 is shown in Table 1.

[0051] Table 1

[0052]

[0053] 4. Use an ultrasonic disruptor to disrupt the bacterial cells in the above liquid. The disruption time is 10 min (30W power, 2s sonication, 5s pause). After disruption, centrifuge at 5000 rpm and 4℃ for 15 min using a low-temperature refrigerated centrifuge. Collect the supernatant for protein purification.

[0054] Protein purification:

[0055] 1. Gently stir the resin a few times to ensure complete suspension. Transfer the resin solution to a gravity flow column using a pipette. Allow it to stand for a period of time to allow the resin to settle, and drain the storage buffer from the tube.

[0056] 2. Equilibrate the column with 10 times the resin volume of Buffer 1. Flow the buffer completely at a rate of 0.5–1 mL / min.

[0057] 3. Add the supernatant after crushing to the column and mix the resin and liquid thoroughly. Allow the target protein to fully bind with the resin at 4°C for 0.5-1 h. After the target protein has fully bound with the resin, slowly elute the waste liquid at a rate of 0.5-1 mL / min.

[0058] 4. Add 5 to 10 column volumes of Buffer 1 to rinse the resin and wash away any impurities and proteins.

[0059] 5. Elute the target protein into a centrifuge tube with 2 column volumes of Buffer 2. The elution should be performed in three stages, and the final products can be combined or stored separately.

[0060] Buffer 2 is composed of Buffer 1 with 3.6031g of maltose added and pH adjusted to 7.4.

[0061] Characterization:

[0062] The SDS-PAGE electrophoresis results of the CsHCT recombinant protein and the PMAL-C5X empty vector protein are as follows: Figure 1 As shown, the size of the CsHCT recombinant protein band after adding the MBP tag is approximately 91 kDa; the size of the PMAL-C5X empty vector protein band is approximately 42 kDa, both within a similar range. Example 2 is used for subsequent in vitro enzyme activity examples.

[0063] The amino acid sequence of the acyltransferase CsHCT is shown in SEQ ID No: 1:

[0064] SEQ ID No: 1

[0065] MRIEVRESTMVKAAQEIPSSKRRLWNSNVDLVVPSFHTPSVYFYRPNGSPNFFEPHVLKDALSRA

[0066] LVAFYPMAGRLARDDDGRIEIDCNDEGVLFVEAESDSCVDDFGDFAPTLELRQLIPAVDYSKGIS

[0067] SYPILVLQVTYFKCGGVSLGVGMQHHVADGSSGLHFVNTWSDIARGLDLTIPPFIDRTLLRARDP

[0068] PQPAFHHIEYQPPPSMKVDPQSTKPESVPEATVSIFKLTKDQLNTLKAKSKEDGNMVNYSSYEML

[0069] AGHVWRSVCKARGLPDNQETKLYIATDGRSRLRPALPPGYFGNVIFTTTPLAIAAGDLQSKPTWY

[0070] AASRIHDALVRMDNDYLRSALDYLELQPDLKALVRGAHTFKCPNLGITSWVRLPIHDADFGWG

[0071] RPIFMGPGGIAYEGLAFALPSPTNDGSLSVAISLQTEHMKLFEKFLFDI

[0072] Example 3: In vitro synthesis of kaempferol-3-O-(6'-coumaryl)-glucoside

[0073] Experimental methods:

[0074] Using p-coumaryl-CoA as the acyl donor and kaempferol-3-O-glucoside as the substrate, in vitro enzyme activity assays were performed. The reaction system consisted of 620 μL of 100 mM sodium phosphate buffer (pH = 8), 20 μM kaempferol-3-O-glucoside substrate, 20 μM acyl donor, and 10 μL (10–20 μg) of purified CsHCT recombinant protein or PMAL-C5X empty vector protein. After incubation at 40 °C for 4 hours, 620 μL of methanol was added to terminate the reaction. The mixture was centrifuged at 12,000 rpm for 15 min at 4 °C, and 500 μL of the supernatant was transferred to a brown liquid chromatography bottle for LC-Q-Tof-MS identification.

[0075] LC-Q-Tof-MS was performed using a time-of-flight LC-MS / MS system in negative ion scanning mode. The scan range was m / z 100-1500, with the following settings: sheath gas temperature, 350℃; nebulizer, 35psi; gas flow rate: 8 L / min; gas temperature, 320℃; sheath gas flow rate: 11 L / min.

[0076] Experimental results:

[0077] like Figure 2 As shown, when using p-coumaryl-CoA as the acyl donor and kaempferol-3-O-glucoside as the substrate, CsHCT catalyzes the formation of a single product peak compared to the empty protein. By comparing the retention time and ion fragmentation information of the product peak and the standard, the product was identified as kaempferol-3-O-(6'-coumaryl)-glucoside, with a molecular ion peak of 593 m / z in negative ion mode.

[0078] The above results indicate that, under in vitro conditions, CsHCT can catalyze the formation of the corresponding kaempferol-3-O-glucoside from p-coumaryl coenzyme A as an acyl donor.

[0079] Example 4: Application of the gene encoding the acyltransferase CsHCT

[0080] 1. The open reading frame of CsHCT was incorporated into the plant binary expression vector pK7FWG2.0 using BP and LR enzymes, with primers shown in Seq 6 and Seq 7, to obtain the recombinant vector CsHCT-PK7. 100 ng of the recombinant plasmid and the empty PK7 plasmid were added to 50 μL of GV3101 strain, slowly mixed, and then subjected to the following incubation cycles: 5 min on ice, 5 min in liquid nitrogen, 5 min in a 37°C water bath, and 5 min on ice. After shaking culture, the bacterial culture was spread onto LB agar containing 100 mg / L spectinomycin (Spec) and 50 mg / L rifampin (Rif), and incubated at 28°C for 48–72 hours. Single colonies were streaked and colony PCR was performed to screen for samples with correct bands. These samples were then stored in 50% sterile glycerol at -80°C.

[0081] SEQ ID No: 6:

[0082] GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGAGGATAGAAGTGAGGGAGTCG

[0083] SEQ ID No: 7:

[0084] GGGGACCACTTTGTACAAGAAAGCTGGGTTATATGTCAAACAAGAACTTCTCAAAGA

[0085] 2. Transfer 100 μL of the preserved bacterial culture to 5 mL of LB liquid containing Spec and Rif, and incubate overnight at 28°C. The next day, scale up the culture at a 1:100 ratio, and incubate at 28°C with a shaker at 200-220 rpm until the OD600 of the bacterial culture reaches 0.6-0.8. Centrifuge at 1000g for 5 min, discard the supernatant, resuspend the bacterial cells twice with an equal volume of Buffer 3, and adjust the OD600 value to 0.8. This culture can then be used for tea seedling injection. The control group was transformed with PMAL-C5X empty vector, and the experimental group was transformed with CsHCT-PMAL.

[0086] The composition of Buffer 3 is shown in Table 2.

[0087] Table 2

[0088]

[0089] 3. Select healthy one-year-old tea seedlings with symmetrical growth on both sides of the leaves as experimental materials. Use a 1mL syringe (without the needle) to draw up the infection solution and inject it into the back of the leaves; one leaf constitutes one replicate. After injection, place the tea seedlings in a culture room at 25℃ with a light / dark cycle of 16h / 8h for 3 days. Take the infected leaf area, flash-freeze it with liquid nitrogen, grind it, and place it in a -80℃ freezer for later use.

[0090] 4. The qRT-PCR primers for the tea plant internal reference gene GAPDH are shown in SEQ ID No: 8 and SEQ ID No: 9. The qRT-PCR primers for CsHCT are shown in SEQ ID No: 10 and SEQ ID No: 11.

[0091] SEQ ID No: 8:

[0092] TTGGCATCGTTGAGGGTCT

[0093] SEQ ID No: 9:

[0094] CAGTGGGAACACGGAAAGC

[0095] SEQ ID No: 10:

[0096] GGAGTGTCCCTCGGTGTTGG

[0097] SEQ ID No: 11:

[0098] GTGGTGGAAAGCAGGTTGTG

[0099] Experimental results:

[0100] like Figure 3 As shown, compared to the control group injected with empty vector, the transcriptional level of CsHCT was significantly upregulated in the experimental group overexpressing CsHCT. Figure 3 Meanwhile, kaempferol-3-O-(6'-coumaryl)-glucoside was extracted from the leaves of tea seedlings in both the control and experimental groups and detected by LC-QQQ-MS. The results showed that the content of kaempferol-3-O-(6'-coumaryl)-glucoside in the experimental group was significantly increased. Figure 4 ).

[0101] This indicates that CsHCT possesses the ability to catalyze the acylation modification of kaempferol-3-O-glucoside in plants, demonstrating certain application prospects in the biosynthesis of acylated kaempferol-3-O-glucoside.

[0102] Through the acyltransferase CsHCT, its encoding gene, and its application in this embodiment, the in vitro enzymatic synthesis of kaempferol-3-O-(6'-coumaryl)-glucoside can be achieved. At the same time, a gene editing method is disclosed to obtain an enzyme capable of catalyzing the synthesis of kaempferol-3-O-(6'-coumaryl)-glucoside, showing certain application prospects in the field of acylated kaempferol-3-O-glucoside biosynthesis.

[0103] The above embodiments are merely exemplary embodiments of this application and are not intended to limit this application. The scope of protection of this application is defined by the claims. Those skilled in the art can make various modifications or equivalent substitutions to this application within its substance and scope of protection, and such modifications or equivalent substitutions should also be considered to fall within the scope of protection of this application.

Claims

1. An application of an acyltransferase CsHCT, characterized in that, Kaempferol-3-O-(6'-coumaryl)-glucoside was synthesized in vitro via the acyltransferase CsHCT catalysis. The amino acid sequence of the acyltransferase CsHCT is shown in SEQ ID No:

1.

2. The application of the acyltransferase CsHCT according to claim 1, characterized in that, Kaempferol-3-O-(6'-coumaryl)-glucoside was synthesized in vitro using the acyltransferase CsHCT catalyzing the reaction of coumaroyl coenzyme A with kaempferol-3-O-glucoside.

3. A method for in vitro synthesis of kaempferol-3-O-(6'-coumaryl)-glucoside, characterized in that, Includes the following steps: Using p-coumaryl coenzyme A as the acyl donor and kaempferol-3-O-glucoside as the acyl acceptor, acyltransferase CsHCT was used as the catalytic enzyme to carry out an enzymatic reaction to obtain kaempferol-3-O-(6'-coumaryl)-glucoside. The amino acid sequence of the acyltransferase CsHCT is shown in SEQ ID No:

1.

4. An application of the gene encoding the acyltransferase CsHCT as described in claim 1, characterized in that, Kaempferol-3-O-(6'-coumaryl)-glucoside was synthesized in vitro from kaempferol-3-O-glucoside via prokaryotic expression of the gene encoding the acyltransferase CsHCT.

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