Acyltransferase CsHCT, coding gene and application thereof

Abstract

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

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, and in particular to an acyltransferase CsHCT, an encoding gene and applications thereof. Background Art

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

[0003] However, in the prior art, separation and purification methods are generally used to extract kaempferol-3-O-(6'-coumaryl)-glucoside from samples. These methods are complex and the process is cumbersome, resulting in low yields and limited research, making it difficult to fully study and utilize this substance. Furthermore, 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, new technologies are urgently needed to provide new ideas for the in vitro synthesis of kaempferol-3-O-(6'-coumaryl)-glucoside. Summary of the Invention

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

[0006] In order to solve the above technical problems, the present invention specifically provides the following technical solutions:

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

[0008] The present invention provides a coding gene for expressing acyltransferase CsHCT.

[0009] The present invention provides a primer pair for amplifying a gene encoding acyltransferase CsHCT, wherein the upstream primer of the primer pair has a nucleotide sequence as shown in SEQ ID NO: 2;

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

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

[0012] As a preferred embodiment of the present invention, the acyltransferase CsHCT is used to catalyze the in vitro synthesis of coumaroyl-CoA and kaempferol-3-O-glucoside to prepare kaempferol-3-O-(6'-coumaroyl)-glucoside.

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

[0014] Kaempferol-3-O-(6'-coumaryl)-glucoside was obtained by enzymatic reaction using p-coumaroyl-CoA as acyl donor, kaempferol-3-O-glucoside as acyl acceptor and acyltransferase CsHCT as catalytic enzyme.

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

[0016] The present invention provides an application of a gene encoding an acyltransferase CsHCT, and realizes the in vitro synthesis of kaempferol-3-O-(6'-coumaryl)-glucoside through prokaryotic expression of the gene encoding the acyltransferase CsHCT.

[0017] The present invention provides a method for synthesizing and preparing acyltransferase CsHCT in vitro, comprising the following steps:

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

[0019] The CsHCT-PMAL recombinant plasmid was chemically transformed into Escherichia coli BL21(DE3) and disrupted using an ultrasonic disruptor. The disruption lasted 10 minutes at 30W power, with 2 seconds of sonication and a 5-second pause. After disruption, the cells were centrifuged at 5000 rpm and 4°C for 15 minutes. The supernatant was collected and purified to obtain the acyltransferase CsHCT.

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

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

[0022] The present invention establishes an in vitro enzymatic activity reaction system for kaempferol-3-O-(6'-coumaryl)-glucoside based on the acyltransferase CsHCT. Under in vitro conditions, the system can catalyze the synthesis of kaempferol-3-O-glucoside from p-coumaroyl-CoA as an acyl donor. The product can be detected by high-performance liquid chromatography (HPLC) and identified by liquid chromatography-tandem mass spectrometry (LC-MS / MS). This provides a strategy for the efficient synthesis of kaempferol-3-O-(6'-coumaryl)-glucoside by in vitro enzymatic methods in the future.

[0023] The present invention provides a gene encoding acyltransferase CsHCT, which is expressed and purified through prokaryotic expression to obtain CsHCT recombinant protein, and further discloses the use of this encoding gene in in vitro synthesis of kaempferol-3-O-(6'-coumaroyl)-glucoside. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the embodiments or the description of the prior art. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other implementation drawings based on the provided drawings without inventive effort.

[0025] Figure 1 The present invention provides a gel image of the target protein purification shown in Example 2;

[0026] Figure 2 The present invention provides the in vitro enzyme activity chemical profile shown in Example 3;

[0027] Figure 3 The present invention provides a bar graph of the transcription level of CsHCT in the experimental group overexpressing CsHCT as shown in Example 4;

[0028] Figure 4 The present invention provides a bar graph of the content of kaempferol-3-O-(6'-coumaryl)-glucoside in the experimental group overexpressing CsHCT as shown in Example 4. DETAILED DESCRIPTION

[0029] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. 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 making creative efforts are within the scope of protection of the present invention.

[0030] Example 1: Gene Editing

[0031] Fresh tea leaves were ground into powder in liquid nitrogen. Total RNA was extracted from tea leaf tissue using a plant RNA extraction kit and treated with DNAase to remove DNA interference. The resulting RNA sample was used as a template for reverse transcription using a kit to synthesize the first strand of cDNA. PCR amplification was performed using KOD plus enzyme. The upstream primer of the primer pair for amplifying the gene encoding 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 running to verify the band position, and the target band was recovered from the gel. Then, it was ligated and transformed into the cloning vector pEASY-Blunt-Zero and sent to Shanghai Bioengineering Co., Ltd. for sequencing.

[0037] This gene fragment is the gene encoding the acyltransferase CsHCT.

[0038] This gene has been previously reported to be involved in lignin synthesis, catalyzing the synthesis of substrates such as QA / SA. The present invention provides the use of this gene for the in vivo and in vitro synthesis of kaempferol-3-O-(6'-coumaroyl)-glucoside. Specifically, this gene encoding kaempferol was expressed and purified via prokaryotic expression to produce the recombinant protein CsHCT, which catalyzes the synthesis of kaempferol-3-O-(6'-coumaroyl)-glucoside.

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

[0040] Recombinant vector construction

[0041] The sequenced CsHCT-peasy-Blunt-Zero vector was used as a template for cloning, and the primers are shown in SEQ ID Nos. 4 and 5. The recovered product was ligated into the PMAL-C5X vector, which had been double-digested with the restriction endonucleases NdeI and BamHI. The product was then transformed into Escherichia coli DH5α using the heat shock method. The bacterial suspension was spread onto LB solid medium containing 100 mg / L ampicillin (Amp) and incubated in an inverted manner at 37°C for 12-14 hours. After spot testing, colony PCR, and verification by testing, the CsHCT-PMAL recombinant plasmid was 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). The plasmids were spread onto solid LB plates containing Ampr resistance and incubated inverted at 37°C for approximately 10 hours. Single colonies from the plates were then streaked and verified by colony PCR. A portion of the bacterial suspension containing the correct colony PCR band was added to 500 μL of liquid medium containing Ampr. The culture was incubated overnight at 37°C with a shaker at 200 rpm. Afterward, 500 μL of 50% sterile glycerol was added and the cells were stored at -80°C until use.

[0048] 2. Select a positive monoclonal strain that has been verified to be Amp-resistant and culture it in 5 mL of liquid LB medium containing Amp resistance. Incubate it in a thermostatted shaker at 200-220 rpm for 10-12 hours at 37°C. Take 200 μL of the cultured bacteria and add it to 200 mL of LB liquid containing the same resistance medium at a ratio of 1:100 for expansion. Incubate it in a thermostatted shaker at 200-220 rpm at 37°C until the OD600 of the culture reaches 0.6-0.8.

[0049] 3. Set aside 2 mL of the bacterial culture. Add the inducer IPTG to the remaining culture medium and induce for 24 hours on a constant temperature shaker at 16°C and 200-220 rpm. Take 2 mL of the induced culture medium. Centrifuge the remaining culture medium in a refrigerated centrifuge at 4000g and 4°C for 15 minutes, removing the supernatant. Collect the induced cells and resuspend them in 20-25 mL of Buffer 1. Collect the resuspended cells 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 cells for 10 minutes (power 30W, ultrasonication for 2 seconds, pause for 5 seconds). After disruption, centrifuge at 5000 rpm, 4°C, for 15 minutes. Collect the supernatant for protein purification.

[0054] Protein purification:

[0055] 1. Gently stir the resin several times to completely suspend it. Use a pipette to transfer the resin solution to a gravity flow column. Let it sit for a while to allow the resin to settle and drain the storage buffer from the tube.

[0056] 2. Equilibrate the column with 10 times the volume of the resin in Buffer 1. Allow the buffer to flow out 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. Allow the target protein to fully bind to the resin at 4°C for 0.5-1 hour. After the target protein is fully bound to the resin, drain the waste liquid slowly at a rate of 0.5-1 mL / min.

[0058] 4. Add 5 to 10 times the column volume of Buffer 1 to rinse the resin and elute the impurities.

[0059] 5. Elute the target protein into a centrifuge tube with 2 column volumes of Buffer 2. The elution is completed in three steps and can be combined or stored separately.

[0060] The specific composition of Buffer 2 is to add 3.6031g maltose to Buffer 1 and adjust the pH to 7.4.

[0061] Characterization:

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

[0063] The amino acid sequence of 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] In vitro enzyme activity assays were performed using p-coumaryl-CoA as the acyl donor and kaempferol-3-O-glucoside as the substrate. 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 protein. After incubation at 40°C for 4 hours, the reaction was terminated by adding 620 μL of methanol. The reaction was centrifuged at 12,000 rpm for 15 minutes at 4°C, and 500 μL of the supernatant was transferred to a brown liquid chromatography vial for analysis by LC-Q-Tof-MS.

[0075] LC-Q-Tof-MS was used for mass spectrometry analysis using a time-of-flight liquid chromatography-mass spectrometer in negative ion scanning mode. The scan range was m / z 100-1500, and the following settings were used: sheath gas temperature, 350°C; nebulizer, 35 psi; gas flow rate, 8 l / min; gas temperature, 320°C; sheath gas flow rate, 11 l / min.

[0076] Experimental results:

[0077] like Figure 2 As shown, using p-coumaroyl-CoA as the acyl donor and kaempferol-3-O-glucoside as the substrate, CsHCT catalyzes the generation of a single product peak compared to empty protein. Comparison of the retention time and ion fragmentation of the product peak with that of the standard confirmed the product to be kaempferol-3-O-(6'-coumaroyl)-glucoside, with a molecular ion peak at 593 m / z in negative ion mode.

[0078] The above results indicate that, under in vitro conditions, CsHCT can catalyze the conversion of kaempferol-3-O-glucoside to the corresponding kaempferol-3-O-(6'-coumaryl)-glucoside using p-coumaroyl-CoA as the acyl donor.

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

[0080] 1. The open reading frame of CsHCT was ligated into the plant binary expression vector pK7FWG2.0 using BP and LR enzymes. The primers are shown in Seq 6 and Seq 7, resulting in the recombinant vector CsHCT-PK7. 100 ng of the recombinant plasmid and the PK7 empty plasmid were added to 50 μL of the GV3101 strain, mixed by gentle pipetting, and incubated on ice for 5 minutes, in liquid nitrogen for 5 minutes, and in a 37°C water bath for 5 minutes. After a 5-minute ice bath, the culture was shaken and plated onto LB solid medium supplemented with 100 mg / L spectinomycin (Spec) and 50 mg / L rifampicin (Rif). The culture was incubated in a 28°C oven for 48–72 hours. Single colonies were streaked and screened for correct bands by colony PCR. These samples were then stored at −80°C in a 50% sterile glycerol freezer.

[0081] SEQ ID No: 6:

[0082] GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGAGGATAGAAGTGAGGGAGTCG

[0083] SEQ ID No: 7:

[0084] GGGGACCACTTTGTACAAGAAAGCTGGGTTATATGTCAAACAAGAACTTCTCAAAGA

[0085] 2. Pipette 100 μL of the preserved bacterial suspension into 5 mL of liquid LB containing Spec and Rif. Incubate overnight in a 28°C incubator. The next day, expand the culture at a 1:100 ratio and incubate on a 28°C shaker at 200-220 rpm until the OD600 reaches 0.6-0.8. Centrifuge at 1000 g for 5 minutes, discard the supernatant, and resuspend the cells twice in equal volumes of Buffer 3, adjusting the OD600 to 0.8. This is ready for tea seedling injection. The control group is transformed with the PMAL-C5X empty vector, while the experimental group is transformed with CsHCT-PMAL.

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

[0087] Table 2

[0088]

[0089] 3. Select one-year-old tea seedlings with healthy growth and consistent growth on both sides of their leaves as experimental materials. Use a 1mL syringe with a removed needle to draw up the infection solution and inject it onto the underside of the leaf. One leaf constitutes one replicate. After injection, incubate the seedlings in a 25°C incubator with a 16h / 8h light / dark cycle for three days. Samples of the infected leaf sections were collected, quickly frozen in liquid nitrogen, ground, and stored in a -80°C freezer until ready for 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 with the control group injected with empty vector, the transcription level of CsHCT in the experimental group overexpressing CsHCT was significantly upregulated ( Figure 3 ), at the same time, kaempferol-3-O-(6'-coumaryl)-glucoside was extracted from the leaves of tea seedlings in the control group and the experimental group, and LC-QQQ-MS detection was performed. The results showed that the content of kaempferol-3-O-(6'-coumaryl)-glucoside in the experimental group increased significantly ( Figure 4 ).

[0101] These results indicate that CsHCT has the function of catalyzing the acylation modification of kaempferol-3-O-glucoside in plants, and shows certain application prospects in the biosynthesis of acylated kaempferol-3-O-glucoside.

[0102] The acyltransferase CsHCT, encoding gene, and application thereof of this embodiment can achieve in vitro enzymatic synthesis of kaempferol-3-O-(6'-coumaryl)-glucoside. A gene editing method is also 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 biosynthesis of acylated kaempferol-3-O-glucoside.

[0103] The above embodiments are merely exemplary embodiments of the present application and are not intended to limit the scope of the present application. The scope of protection of the present application is defined by the claims. Those skilled in the art may make various modifications or equivalent substitutions to the present application within the essence and scope of protection of the present application, and such modifications or equivalent substitutions shall also be deemed to fall within the scope of protection of the present application.

Claims

1. An acyltransferase CsHCT, characterized in that: The amino acid sequence of the acyltransferase CsHCT is shown in SEQ ID No:

1.

2. A use of the acyltransferase CsHCT as claimed in claim 1, characterized in that: The in vitro synthesis of kaempferol-3-O-(6'-coumaryl)-glucoside was catalyzed by the acyltransferase CsHCT.

3. The use of acyltransferase CsHCT according to claim 2, characterized in that: The acyltransferase CsHCT is used to catalyze the in vitro synthesis of coumaroyl coenzyme A and kaempferol-3-O-glucoside to prepare kaempferol-3-O-(6'-coumaroyl)-glucoside.

4. A method for synthesizing kaempferol-3-O-(6'-coumaryl)-glucoside in vitro, characterized in that: The following steps are involved: Using p-coumaroyl-CoA as an acyl donor, kaempferol-3-O-glucoside as an acyl acceptor, and acyltransferase CsHCT as a catalytic enzyme, an enzyme-catalyzed reaction is carried out to obtain kaempferol-3-O-(6'-coumaroyl)-glucoside; The amino acid sequence of the acyltransferase CsHCT is shown in SEQ ID No:

1.

5. A gene encoding the acyltransferase CsHCT according to claim 1.

6. A primer pair for amplifying the gene encoding the acyltransferase CsHCT according to claim 2, characterized in that: The nucleotide sequence of the upstream primer of the primer pair is shown in SEQ ID NO: 2; The nucleotide sequence of the downstream primer of the primer pair is shown in SEQ ID NO:

3.

7. A use of the gene encoding acyltransferase CsHCT as claimed in claim 5, characterized in that: The in vitro synthesis of kaempferol-3-O-(6'-coumaryl)-glucoside is achieved by prokaryotic expression of the coding gene of the acyltransferase CsHCT.

8. A method for preparing acyltransferase CsHCT by in vitro synthesis, according to claim 7, characterized in that: The steps include: The correctly sequenced CsHCT-peasy-Blunt-Zero vector was used as a template for cloning, and the recovered product was ligated to the PMAL-C5X vector double-digested with restriction endonucleases NdeI and BamHI, and transformed into Escherichia coli DH5α by heat shock method to obtain the CsHCT-PMAL recombinant plasmid; The CsHCT-PMAL recombinant plasmid was transferred into Escherichia coli BL21 (DE3) by chemical transformation, and the bacteria were broken by ultrasonic disruptor. The breaking time was 10 min, the power was 30 W, the ultrasound was 2 s, and the pause time was 5 s. After the breaking was completed, the supernatant was collected and purified to obtain the acyltransferase CsHCT.

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