Lycium barbarum phenolic amide derivative acyltransferase as well as coding gene and application thereof

Abstract

The invention discloses lycium barbarum phenolic amide derivative acyltransferase as well as a coding gene and application thereof. The amino acid sequence of the lycium barbarum phenolic amide derivative acyltransferase is any one of a sequence 5 to a sequence 8. According to the invention, four phenolic amide acyltransferase genes are cloned from a lycium plant, can catalyze hydroxycinnamyl coenzyme A to be transferred to an N atom of polyamine to be condensed to generate a corresponding HCAAs component, and have important application value in in-vitro biosynthesis of a lycium plant HCAAs derivative; or the method has a reference effect and important significance on in-vitro catalytic synthesis of phenolic amide derivatives from other species sources.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of plant genetic engineering and biotechnology, and particularly relates to a group of coding genes of phenolic amide derivative acyltransferases and application of proteins thereof. BACKGROUND

[0002] Lycium L. plants, and was first recorded in Shennong Bencao Jing, with a medicinal history of more than 2,000 years. It is a traditional Chinese precious medicinal material and one of the substances of food and medicine. The polysaccharides, organic acids, flavonoids, carotenoids and polyphenols contained in Lycium barbarum are considered to be the main chemical substances in Lycium barbarum and the basis for exerting pharmacological activities. Modern pharmacological studies have shown that Lycium barbarum has antibacterial, anti-inflammatory, antioxidant, anti-aging, anti-tumor, neuroprotective, immunomodulatory and liver-protecting and vision-improving effects, and is one of the natural sources of drugs for treating various diseases.

[0003] Hydroxycinnamic acid amides (HCAAs), also known as phenolic amides, are a class of important secondary metabolites widely existing in higher plants, and are usually present in the fruits and root barks of plants as major phenolic compounds. HCAAs are a class of mono-, di- or tri-substituted amide derivatives with obvious structural characteristics, which are formed by covalently linking hydroxycinnamic acid and its derivatives (such as p-coumaric acid, caffeic acid and ferulic acid) to aromatic monoamines or aliphatic polyamines (such as tyramine, putrescine, spermidine and spermine) through amide bonds. Dicaffeoyl polyamine derivatives (DCPAds) are a class of characteristic HCAAs compounds mainly contained in Lycium barbarum, including dicaffeoyl spermidine compounds and dicaffeoyl spermine compounds. DCPAds mainly take spermidine or spermine as the skeleton, and are composed of 2 molecules of caffeic acid or dihydrocaffeic acid through amide bonds, and are relatively rare in nature, and most exist in Lycium L. plants. DCPAds compounds can improve the short-term learning and memory ability of transgenic fruit flies, and may play a role in the treatment of Alzheimer's disease. In addition, such compounds also have the effects of treating parasitic trypanosomiasis, resisting sepsis, lowering blood pressure, lowering lipid, resisting cancer and resisting aging, and can promote the fermentation production of the anti-tumor drug pingyangmycin. Therefore, DCPAds have broad application prospects in the fields of medicine, food and nutrition and health care. However, there are few studies on the metabolic pathway of such compounds at present.

[0004] HCAAs in plants are mainly hydroxycinnamoyl-CoA esters and biogenic amines condensed under the action of hydroxycinnamoyl transferase, and the reaction plays an important role in the synthesis, transport and storage of plant secondary metabolites. The genus Lycium is a rich natural source of HCAAs, and the research on HCAAs in Lycium is mainly focused on the separation, extraction and structural identification of such substances, and there are few reports on the metabolic pathway of DCPAds or HCAAs.

[0005] HCAAs in the genus Lycium have important application prospects and potential economic value in the treatment of Parkinson's disease, Alzheimer's disease and cardiovascular disease. Therefore, it is of great value to carry out research on the coding genes and proteins of the key acyltransferase in the biosynthetic pathway of HCAAs in the genus Lycium by using the genetic resources of the genus Lycium and biological technology. SUMMARY

[0006] The present application provides a protein, which is any one of (a1)-(a8) as follows:

[0007] (a1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 5;

[0008] (a2) a protein derived from (a1) by substitution and / or deletion and / or addition of one or several amino acid residues and having phenolic amide derivative acyltransferase;

[0009] (a3) a protein consisting of the amino acid sequence shown in SEQ ID NO: 6;

[0010] (a4) a protein derived from (a3) by substitution and / or deletion and / or addition of one or several amino acid residues and having phenolic amide derivative acyltransferase;

[0011] (a5) a protein consisting of the amino acid sequence shown in SEQ ID NO: 7;

[0012] (a6) a protein derived from (a5) by substitution and / or deletion and / or addition of one or several amino acid residues and having phenolic amide derivative acyltransferase;

[0013] (a7) a protein consisting of the amino acid sequence shown in SEQ ID NO: 8;

[0014] (a8) a protein derived from (a7) by substitution and / or deletion and / or addition of one or several amino acid residues and having phenolic amide derivative acyltransferase.

[0015] The present application also provides related biological materials of the protein, which are any one of the following:

[0016] C1) The nucleic acid molecule that encodes the protein;

[0017] C2) An expression cassette containing the nucleic acid molecule described in C1);

[0018] C3) A recombinant vector containing the nucleic acid molecule described in C1), or a recombinant vector containing the expression cassette described in C2);

[0019] C4) Recombinant microorganisms containing the nucleic acid molecules described in C1), or recombinant microorganisms containing the expression cassette described in C2), or recombinant microorganisms containing the recombinant vector described in C3).

[0020] Furthermore, the nucleic acid molecule is any of the following:

[0021] B1) The DNA molecule shown in SEQ ID No. 1;

[0022] B2) A DNA molecule that hybridizes under stringent conditions with the DNA molecule defined in (B1) and encodes the protein thereon;

[0023] B3) has a DNA sequence that is more than 75% identical to that of B1) and encodes the protein therein;

[0024] B4) The DNA molecule shown in SEQ ID No. 2;

[0025] B5) A DNA molecule that hybridizes under stringent conditions with the DNA molecule defined in (B4) and encodes the protein thereon;

[0026] The DNA molecule that has more than 75% DNA sequence identity with B4) and encodes the protein described therein;

[0027] B7) The DNA molecule shown in SEQ ID No. 3;

[0028] B8) A DNA molecule that hybridizes under stringent conditions with the DNA molecule defined in (B7) and encodes the protein thereon;

[0029] DNA molecules that have more than 75% DNA sequence identity with B7) and encode the protein described therein;

[0030] The DNA molecule shown in SEQ ID No. 4 (B10);

[0031] B11) A DNA molecule that hybridizes under stringent conditions with a DNA molecule defined by (B10) and encodes the protein thereon;

[0032] The DNA sequence of B12) and B10) has more than 75% identity and encodes the DNA molecule of the protein.

[0033] The use of the above-mentioned protein or the above-mentioned related biological material in the synthesis of phenolic amide derivatives should also be within the protection scope of the present application.

[0034] Further, the phenolic amide derivative acyltransferase catalyzes the synthesis of phenolic amide derivatives from polyamines and hydroxycinnamoyl-CoA.

[0035] Further, the phenolic amide acyltransferase synthesizes N,N'-dicaffeoyl spermidine, N,N'-dicafeoyl spermine, N,N'-dicaffeoyl spermine, N,N'-dicafeoyl spermine or N,N',N"-tricaffeoyl spermine.

[0036] The present application also provides a method for preparing phenolic amide derivatives, comprising the step of using the above-mentioned protein as phenolic amide derivative acyltransferase for enzymatic reaction.

[0037] Further, the method comprises the step of using the above-mentioned protein as phenolic amide derivative acyltransferase for catalyzing the synthesis of N,N'-dicaffeoyl spermidine, N,N'-dicafeoyl spermine and N,N',N"-tricaffeoyl spermine from spermine.

[0038] Further, the method comprises the step of using the above-mentioned protein as phenolic amide derivative acyltransferase for catalyzing the synthesis of N,N'-dicaffeoyl spermidine and N,N'-dicafeoyl spermine from spermidine.

[0039] The present application clones four phenolic amide derivative acyltransferase genes from Lycium genus plants, which can catalyze the condensation of hydroxycinnamoyl-CoA (coumaroyl-CoA and caffeoyl-CoA) to the N atom of polyamines (spermidine and spermine) to generate corresponding HCAAs components, which has important application value in the in vitro biosynthesis of HCAAs derivatives of Lycium genus plants, and also has reference role and important significance for the in vitro catalytic synthesis of phenolic amide derivatives from other species. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 .LaSHT1, LaSHT2, LcSHT3 and LcSHT4 recombinant purified protein SDS-PAGE electrophoresis map. M: protein Marker; T: total protein of supernatant after induction; lanes 1-4 are recombinant proteins eluted at 50, 100, 150 and 200 mM imidazole concentrations, respectively; lane 5 is pET32a empty vector; the left arrow indicates the target protein.

[0041] Figure 2Synthetic route of N,N'-dicaffeoyl spermine P1 and N,N',N"-tricaffeoyl spermine P5 generated by SHT recombinant purified protein in vitro catalyzing spermine and caffeoyl-CoA (A), LC spectrum of SHT recombinant protein in vitro enzymatic reaction (B), (C), (D), (E) and (F) are the secondary mass spectrum of LaSHT1, LaSHT2, LcSHT3 and LcSHT4 enzymatic reaction products, respectively.

[0042] Figure 3 Synthetic route of N,N'-dicaffeoyl spermidine P2 generated by SHT recombinant purified protein in vitro catalyzing spermidine and caffeoyl-CoA (A), LC spectrum of SHT recombinant protein in vitro enzymatic reaction (B), (C), (D) and (E) are the secondary mass spectrum of LaSHT2, LcSHT3 and LcSHT4 enzymatic reaction products, respectively.

[0043] Figure 4 Synthetic route of N,N'-dicoumaroyl spermine P3 generated by SHT recombinant purified protein in vitro catalyzing spermine and coumaroyl-CoA (A), LC spectrum of SHT recombinant protein in vitro enzymatic reaction (B), (C), (D), (E) and (F) are the secondary mass spectrum of LaSHT1, LaSHT2, LcSHT3 and LcSHT4 enzymatic reaction products, respectively.

[0044] Figure 5 Synthetic route of N1,N'-dicoumaroyl spermidine P4 generated by SHT recombinant purified protein in vitro catalyzing spermidine and coumaroyl-CoA (A), LC spectrum of SHT recombinant protein in vitro enzymatic reaction (B), (C) and (D) are the secondary mass spectrum of LaSHT2 and LcSHT4 enzymatic reaction products, respectively, (E) is the secondary mass spectrum of N1,N'-dicoumaroyl spermidine standard. 10 10

[0045] Figure 6 The results of the enzymatic reaction condition optimization experiment of LcSHT4 protein in Example 9 of the present application. DETAILED DESCRIPTION

[0046] The present application will be further described in conjunction with the specific embodiments, and the examples provided below are only for the purpose of illustrating the present application, and are not intended to limit the scope of the present application. The examples provided below can serve as a guide for further improvement by those skilled in the art, and do not constitute any limitation on the present application.

[0047] ​​The experimental methods in the following examples are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.

[0048] The following examples are more detailed descriptions of the present application, rather than limitations of the present application.

[0049] The Lycium amarum in the following examples is disclosed in the article “Xie D M, Zhang X B, Qian D, et al. Lycium amarum sp. nov. (Solanaceae) from Xizang, supported from morphological characters and phylogenetic analysis [J]. Nordic Journal of Botany, 2016, 34(5): 538-544.”; and the Lycium chinense var. potaninii is disclosed in the article “Qian D, Chen J, Lai C, et al. Dicaffeoyl polyamine derivatives from bitter goji: Contribution to the bitter taste of fruit [J]. Fitoterapia, 2020, 143: 104543.”

[0050] The experimental methods not specifically mentioned in the following examples can be carried out according to conventional methods.

[0051] Example 1: Screening of phenolic amide derivative acyltransferase genes

[0052] The transcriptome data was locally built using Bioedit, and the conserved sequences “HXXXD” and “DFGWG” of the plant secondary metabolism BAHD acyltransferase family were used as bait sequences to search in the transcriptome database of the Lycium plants. The fragments with high sequence similarity were screened as candidate acyltransferase gene sequences, and were named as LaSHT1, LaSHT2, LcSHT3 and LcSHT4 (hereinafter can be referred to as SHT1, SHT2, SHT3 and SHT4).

[0053] Example 2: Cloning of phenolic amide derivative acyltransferase genes

[0054] Total RNA of mature fruit samples of Lycium amarum and Lycium chinense var. potaninii was extracted using RNA Easy Fast Plant Tissue Kit (TIANGEN) kit, and DNase I digestion was performed during the extraction process to ensure the purity of the RNA for subsequent experiments. Genomic DNA removal and RNA reverse transcription were performed using FastKing RT Kit (With gDNase) (TIANGEN) kit. The genomic DNA removal system was as follows: 5×gDNA Buffer 2.0 μL, Total RNA 50 ng-2 μg, and RNase-Free ddH2O was added to 10 μL; incubated at 42°C for 3 min, and then placed on ice. The RNA reverse transcription system was as follows: 10×King RT Buffer 2.0 μL, FastKing RT Enzyme Mix 1.0 μL, FQ-RT Primer Mix 2.0 μL, and RNase-Free ddH2O was added to 10 μL. The reverse transcription system mixed solution was moved to the reaction solution of the DNA removal system and mixed uniformly, incubated at 42°C for 15 min, incubated at 95°C for 3 min, and then placed on ice. The obtained solution was the cDNA of the goji samples in this study, which was stored at -20°C for standby use.

[0055] The full-length of the coding region (CDS) of the candidate phenolic amide derivative acyltransferase gene was cloned using fruit cDNA as a template. The specific sequence information of the primer pair is shown in Table 1. PCR amplification was performed using 2×TransStart FastPfu PCR SuperMix (full type gold), and the amplification system was as follows: 2×TransStart FastPfu PCR SuperMix 12.5 μL, cDNA template 2.0 μL, 10 μM concentration of forward and reverse primers 2.0 μL each, and ddH2O was added to 25 μL. The amplification reaction conditions were as follows: 98°C pre-denaturation for 30 s; 98°C denaturation for 10 s, 55°C annealing for 10 s, 72°C extension for 80 s, 40 cycles; 72°C supplementary extension for 7 min; 4°C holding. The PCR amplification product was identified using 1.0% agarose gel electrophoresis, and then the PCR product was gel recovery purified using GeneJET Gel Extraction Kit (Thermo Scientific) and the concentration of the purified sample was determined.

[0056] The target gene was ligated and transformed using the Lethal Based Fast Cloning Kit (TIANGEN) kit. The ligation reaction system was as follows: target fragment X μL, 2 x Reaction Solution 5.0 μL, pLB Vector (35 ng / μL) 1.0 μL, T4 DNA Ligase (3 U / μL) 1.0 μL, ddH2O to 10 μL. After 5 min of room temperature reaction, it was placed on ice or stored at -20°C.

[0057] 2 μL of the ligation product was aspirated into E. coli Trans 10 competent cells for transformation, and the bacterial liquid after transformation was spread on LB solid culture plates containing ampicillin (Amp) and incubated in a 37°C constant temperature incubator for 12-16 hours. The next day, single colonies were picked and PCR identification was performed using the cloning vector primer to confirm whether the inserted fragment was correct. The PCR reaction system was as follows: 2 x Easy Taq PCR SuperMix 5.0 μL, forward and reverse primers (10 μM) 0.2 μL each, bacterial liquid template 1.0 μL; ddH2O to 10 μL. The PCR amplification program was as follows: 98°C pre-denaturation for 30 s; 98°C denaturation for 10 s, 55°C annealing for 10 s, 72°C extension for 80 s, 30 cycles; 72°C supplementary extension for 7 min; 4°C holding.

[0058] Three positive colonies of each gene were selected after bacterial liquid PCR verification, and were sent to Beijing Norsen Genomic Research Center Co., Ltd. for sequencing. After sequencing analysis, the nucleotide sequence of the phenolic amide derivative acyltransferase gene LaSHT1 obtained by cloning is shown in SEQ ID NO. 1, which contains 1335 bases, and the encoded protein has 444 amino acid residues, and the specific amino acid sequence is shown in SEQ ID NO. 5. The nucleotide sequence of the phenolic amide derivative acyltransferase gene LaSHT2 is shown in SEQ ID NO. 2, which contains 1338 bases, and the encoded protein has 445 amino acid residues, and the specific amino acid sequence is shown in SEQ ID NO. 6. The nucleotide sequence of the phenolic amide derivative acyltransferase gene LcSHT3 is shown in SEQ ID NO. 3, which contains 1344 bases, and the encoded protein has 447 amino acid residues, and the specific amino acid sequence is shown in SEQ ID NO. 7. The nucleotide sequence of the phenolic amide derivative acyltransferase gene LcSHT4 is shown in SEQ ID NO. 4, which contains 1359 bases, and the encoded protein has 452 amino acid residues, and the specific amino acid sequence is shown in SEQ ID NO. 8.

[0059] Table 1. Primer sequences for cloning the full-length of the phenolic amide derivative acyltransferase gene of wolfberry

[0060]

[0061] Example 3: Construction of recombinant expression vector pET32a-SHT

[0062] The primers for the four acyltransferase genes were designed, and the primer information is shown in Table 2. The bacterial liquid after sequencing analysis of the positive clone was used as a template, and the primers shown in Table 2 were used for PCR amplification to obtain the target gene. The PCR amplification system was: 2x TransStart FastPfu PCR SuperMix 25 μL, bacterial liquid template 4.0 μL, 10 μM concentration of forward and reverse primers 2.5 μL each, and ddH2O to 50 μL. The PCR amplification reaction conditions were: 98°C pre-denaturation for 30 s; 98°C denaturation for 10 s, 55°C annealing for 10 s, 72°C extension for 80 s, 40 cycles; 72°C supplementary extension for 7 min; 4°C keeping.

[0063] Meanwhile, the pET32a vector was digested (using NEB restriction endonuclease), and the enzyme reaction system was: CutSmart Buffer 5.0 μL, pET32a plasmid 1.8 μg, BamHI-HF 1.0 μL, and ddH2O to 50 μL. After PCR amplification product and enzyme digestion product were identified by 1.0% agarose gel electrophoresis, they were purified by cutting gel recovery using GeneJET Gel Extraction Kit (Thermo Scientific) and the concentration of the purified sample was measured.

[0064] The acyltransferase target gene was seamlessly spliced into the digested pET32a vector using the pEASY-Basic Seamless Cloning and Assembly Kit reagent kit (Quigen), and the recombination product was transformed into E. coli Trans1-T1 competent cells for transformation. The bacterial liquid was identified by PCR amplification and sent to the company for sequencing to determine the sequence accuracy again. After sequencing correctly, the plasmid was extracted to obtain the recombinant plasmid containing the acyltransferase genes LaSHT1, LaSHT2, LcSHT3 and LcSHT4.

[0065] Table 2. Recombinant plasmid cloning primer sequence information

[0066]

[0067] Example 4: Induction expression and purification of recombinant target protein

[0068] The recombinant positive plasmid containing acyltransferase genes LaSHT1, LaSHT2, LcSHT3 and LcSHT4 obtained in Example 3 was transformed into E. coli Transetta (DE3) expression competent cells, respectively, and the competent cells transformed with empty vector pET32a were used as a control group. Three single colonies of each gene were picked into 1 mL of LB liquid medium containing Amp antibiotic and cultured at 37°C, 200 rpm for 2-3 h. After the bacterial cells became turbid, bacterial liquid PCR was performed for verification.

[0069] 100 μL of positive colonies were taken into 10 mL of LB liquid medium containing Amp antibiotic and cultured at 37°C, 200 rpm overnight. The next day, the overnight bacteria were inoculated into fresh LB liquid medium containing Amp at a dilution ratio of 1:100 and cultured at 37°C, 200 rpm for 2-3 h until the OD value was about 0.4-0.8. After adding an inducer IPTG at a final concentration of 0.2 mM, the culture was continued at 18°C, 200 rpm for 24 h.

[0070] The bacterial cells were collected by low-temperature centrifugation, and the supernatant was discarded to obtain crude protein. The bacterial cells were resuspended in 6 mL of lysis buffer (100 mM Tris-HCl, pH = 7.5, containing 5 mM DTT, 1 mM PMSF and 2.5 mM MgCl2), and the resuspension was incubated in ice water and subjected to ultrasonic disruption (30% power, vibration for 5 s, stop for 5 s, for a total of 30 min). After disruption, the mixture was centrifuged at 4°C, 9000 rpm for 30 min, and the supernatant was collected to obtain total protein T after induction.

[0071] The total protein after induction was subjected to nickel ion affinity chromatography column (Ni-NTA) purification. The specific steps are as follows:

[0072] 50% Ni filler 4 mL was taken into 10 mL empty column, and after standing for a period of time, the excess liquid was drained. 10 column volumes of ddH2O were added to wash the protein purification column;

[0073] 1 column volume of 20 mM imidazole (pH = 7.3) was added to balance the protein purification column;

[0074] The total protein T was added to the purification column containing Ni filler, and the protein was combined with the filler by shaking at 4°C, 80 rpm for 40 min;

[0075] The protein purification column was allowed to stand for 10 min, and the protein solution was collected and added to the purification column again. After standing for 10 min, the filtrate was discarded;

[0076] A low concentration of 20 mM imidazole (pH = 7.3) was added to elute the impure protein, and the filtrate was discarded;

[0077] The target protein was eluted with imidazole containing 50 mM, 100 mM, 150 mM and 200 mM (pH=7.3), and the filtrate was collected into a 1.5 mL sterile centrifuge tube.

[0078] Add the collected protein solution to a 10K ultrafiltration centrifuge tube, centrifuge at 5000 rpm for 10 min at 4 °C, add 5 ml of 100 mM Tris-HCl buffer (pH = 7.5) and centrifuge for 20 min, finally add 5 mL of 100 mM Tris-HCl buffer (pH = 7.5, containing 5 mM DTT and 2.5 mM MgCl2) and centrifuge to concentrate to about 1 mL;

[0079] The concentrated and desalted target protein was collected, and its concentration was determined using a Bradford protein assay kit (Beyotime). 200 μL was aliquoted into 1.5 mL sterile centrifuge tubes and stored at -80°C for later use. This yielded purified proteins of the phenolamide derivative acyltransferases LaSHT1, LaSHT2, LcSHT3, and LcSHT4. The results were then analyzed by polyacrylamide gel electrophoresis (SDS-PAGE).

[0080] Protein gels were prepared using a 10% PAGE high-resolution gel ultra-rapid preparation kit (Servicebio).

[0081] Take 40 μL of protein sample, add 10 μL of 5× protein loading buffer, and heat in a 100℃ water bath for 5 min to denature the protein.

[0082] Remove the sample and let it cool to room temperature. Centrifuge at 12,000 rpm for 3 min at 4℃. Add 10 μL of sample to the gel well and electrophoresis at 200V for 30 min. Then stop the electrophoresis.

[0083] After electrophoresis, remove the gel and place it in Coomassie Brilliant Blue R250 staining solution, and stain on a shaker for 2-3 hours;

[0084] After staining, the gel is placed in the decolorizing solution and decolorized overnight on a shaker;

[0085] The purification effects of phenolamide derivative acyltransferases LaSHT1, LaSHT2, LcSHT3, and LcSHT4 from Lycium chinense were as follows: Figure 1 As shown; from Figure 1 It can be seen that the four recombinant proteins were successfully expressed in Escherichia coli. Recombinant proteins of about 70 kDa were detected in the total protein of the supernatant and in different concentrations of imidazole eluent, and were mainly enriched in the 100 mM and 150 mM imidazole eluent fractions.

[0086] Example 5: Analysis of enzymatic reaction of SHT proteins using caffeoyl-CoA as donor and spermine as acceptor The purified acyltransferases LaSHT1, LaSHT2, LcSHT3 and LcSHT4 obtained in Example 4 were used for biosynthesis. Each 200 μL reaction system contained 120 μM caffeoyl-CoA, 2.5 mM spermine, 100 mM Tris-HCl buffer (pH = 9, containing 10 mM EDTA, pH = 8) and 50 μg purified protein. After reaction at 35 °C for 2 h, the reaction was terminated by 100 μL acetonitrile containing 1% HC1. The reaction sample was centrifuged at 12000 g for 5 min, and the supernatant was filtered through a 0.22 μm microporous membrane and used as the sample for LC-MS analysis. 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) were used as mobile phase for gradient elution (0-5 min, 2%-20% B; 5-15 min, 20%-50% B; 15-15.5 min, 50%-95% B; 15.5-18 min, 95% B; 18-18.5 min, 95%-2% B; 18.5-22 min, 2% B). The flow rate was 0.3 mL·min -1 , the injection volume was 2 μL, and the column temperature was 40 °C.

[0087] LC-MS analysis showed that LaSHT1, LaSHT2 and LcSHT4 had a single peak at a retention time of 4.3 min, as shown in Figure 2 -B. The new compound P1 was identified by mass spectrometry. The secondary mass spectrum of P1 showed a molecular ion peak at m / z 527 + , as shown in Figure 2 -C, 2-D and 2-F. It was shown that the molecular weight of the compound was 526, m / z 163 was the fragment ion of caffeoyloxy, and there were secondary mass spectrum fragments m / z 365, 291, and 220, etc. The fragmentation pattern was consistent with the literature data, and therefore P1 was determined to be N,N'-dicaffeoyl spermine. LcSHT3 had a single peak at a retention time of 7.0 min, as shown in Figure 2 -B. Compound P5 was identified by mass spectrometry. The secondary mass spectrum of P5 showed a molecular ion peak at m / z 689 + , as shown in Figure 2 -E, and the secondary mass spectrum showed characteristic ion fragments m / z 527.2880, 365.2551, 220.0974 and 163.0391 (caffeoyloxy), etc. According to the fragmentation fragments, it was speculated that P5 was N,N'N"-tricaffeoyl spermine.

[0088] The results show that LaSHT1, LaSHT2 and LcSHT4 can catalyze the transfer of caffeoyl-CoA to spermine to generate twice acylated HCAAs compound (N,N'-dicaffeoyl spermine), and LcSHT3 can catalyze the transfer of caffeoyl-CoA to spermine to generate three acylated HCAAs compound (N,N'N"-tricaffeoyl spermine).

[0089] Example 6: Analysis of enzymatic reaction of SHT protein with caffeoyl-CoA as donor and spermidine as acceptor

[0090] The purified acyltransferases LaSHT1, LaSHT2, LcSHT3 and LcSHT4 obtained in Example 4 were used for biosynthesis, and each 200 μL reaction system included: 120 μM caffeoyl-CoA, 2.5 mM spermidine, 100 mM Tris-HCl buffer (pH=9, containing 10 mM EDTA, pH=8) and 50 μg purified protein. The rest of the reaction conditions and LC-MS method were the same as in Example 5.

[0091] After LC-MS analysis, it was found that when spermidine and caffeoyl-CoA were used as substrates, no obvious product signal peak was detected in the chromatogram of LaSHT1, and LaSHT2, LcSHT3 and LcSHT4 had an obvious single peak at a retention time of 5.7 min, obtaining a new compound P2, as shown in Figure 3 The mass spectrum of compound P2 was identified, and its molecular ion peak [M+H] + was shown as m / z 470, containing secondary mass spectrum fragments (m / z 308 (N-caffeoyl spermidine), 220 (acylation reaction characteristic fragment ion) and 163 (caffeoyl group). Figure 3 By comparing with the literature mass spectrum data, its fragmentation fragments were consistent with N,N'-dicaffeoyl spermidine, so P2 was determined to be N,N'-dicaffeoyl spermidine.

[0092] The results show that when spermidine and caffeoyl-CoA are used as substrates, LaSHT2, LcSHT3 and LcSHT4 can catalyze the generation of twice acylated N,N'-dicaffeoyl spermidine.

[0093] Example 7: Analysis of enzymatic reaction of SHT protein with p-coumaroyl-CoA as donor and spermine as acceptor

[0094] The purified acyltransferases LaSHT1, LaSHT2, LcSHT3 and LcSHT4 obtained in Example 4 were used for biosynthesis, each 200 μL reaction system comprising: 120 μM p-coumaroyl-CoA, 2.5 mM spermidine, 100 mM Tris-HCl buffer (pH=9, containing 10 mM EDTA, pH=8) and 50 μg purified protein. The rest of the reaction conditions and LC-MS method were the same as in Example 5.

[0095] It was found by LC-MS analysis that LaSHT1, LaSHT2, LcSHT3 and LcSHT4 could all catalyze to obtain a new compound P3 using spermidine and coumaroyl-CoA as substrates, as shown in formula Figure 4 -B. Compound P3 was identified by mass spectrometry, and the molecular ion peak [M+H] + of P3 was m / z 495, indicating that the molecular weight of the compound was 494, and the fragment ions of m / z 349, 275, 204 and 147 (coumaroyloxy) were secondary mass spectrometry, and the fragmentation mode was consistent with the literature data, so it was determined that P3 was N,N'-d-coumaroyl spermidine.

[0096] The results showed that LaSHT1, LaSHT2, LcSHT3 and LcSHT4 could all catalyze the reaction of coumaroyl-CoA and spermidine to generate a diacylated HCAA compound (N,N'-d-coumaroyl spermidine).

[0097] Example 8: Enzymatic reaction analysis of SHT protein using p-coumaroyl-CoA as donor and spermidine as acceptor

[0098] The purified acyltransferases LaSHT1, LaSHT2, LcSHT3 and LcSHT4 obtained in Example 4 were used for biosynthesis, each 200 μL reaction system comprising: 120 μM p-coumaroyl-CoA, 2.5 mM spermidine, 100 mM Tris-HCl buffer (pH=9, containing 10 mM EDTA, pH=8) and 50 μg purified protein. The rest of the reaction conditions and LC-MS method were the same as in Example 5.

[0099] After LC-MS analysis, it was found that only the ion peaks of LaSHT2 and LcSHT4 were observed around 8 min in the chromatogram Figure 6 .8 min, indicating that they could catalyze spermidine and coumaroyl-CoA to obtain a new compound P4, as shown in formula Figure 5 -B. Compound P4 was identified by mass spectrometry, and the [M+H] + of P4 was m / z 438, indicating that the molecular weight of the compound was 437, and m / z 292, 204 and 147 (coumaroyl) were secondary mass spectrometry fragment ions. By comparison with the standard and literature mass spectrometry data, the fragmentation mode and retention time were consistent withFigure 5 - standard (N1, N 10 - the secondary fragments of coumaroyl spermidine were consistent with P4, so P4 was determined as N1, N 10 - coumaroyl spermidine.

[0100] The results show that LaSHT2 and LcSHT4 can catalyze the transfer of coumaroyl CoA to spermidine to generate a double acylated HCAA compound (N1, N 10 - coumaroyl spermidine).

[0101] Example 9: Optimization of in vitro enzymatic reaction conditions

[0102] In this example, the LcSHT4 protein with the highest protein activity was selected as an example to optimize the enzymatic reaction conditions to obtain the optimal reaction conditions for more enzymatic reaction product amounts.

[0103] Reaction PH: 50mM NaH2PO4-Na2HPO4(5.0-8.0), 100mM Tris-HCl(7.0-10.0, containing 10mM EDTA, pH=8), 50mM Glycine-NaOH(9.0-11.0)

[0104] Reaction temperature: 25-55℃

[0105] Reaction time: 0-150min

[0106] Metal ions: Na + , K + , Mg + , Ca + and EDTA

[0107] The results are shown in Figure 6 From Figure 6 the results, it can be seen that PH 9.0, 35℃ and 120min are the optimal PH, reaction temperature and reaction time for the LcSHT4 enzymatic reaction system, respectively, and LcSHT4 is a metal ion-independent protein type.

[0108] In summary, the optimal reaction conditions for LcSHT4 protein in vitro enzymatic reaction are: 100mM Tris-HCl(pH=9, containing 10mM EDTA, pH=8), 35℃ reaction for 120min.

[0109] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A protein, characterized in that, The protein is any one of (a1)-(a8): (a1) A protein consisting of the amino acid sequence shown in sequence 5; (a2) A protein derived from (a1) by substitution and / or deletion and / or addition of one or more amino acid residues and having a phenolamide derivative acyltransferase. (a3) A protein consisting of the amino acid sequence shown in sequence 6; (a4) Proteins derived from (a3) ​​by substitution and / or deletion and / or addition of one or more amino acid residues and having phenolamide derivative acyltransferases. (a5) A protein consisting of the amino acid sequence shown in sequence 7; (a6) A protein derived from (a5) by substitution and / or deletion and / or addition of one or more amino acid residues and having a phenolamide derivative acyltransferase. (a7) A protein consisting of the amino acid sequence shown in sequence 8; (a8) A protein derived from (a7) by substitution and / or deletion and / or addition of one or more amino acid residues and having a phenolamide derivative acyltransferase.

2. A biomaterial related to the protein of claim 1, characterized in that, The relevant biomaterial is any one of the following: C1) A nucleic acid molecule encoding the protein described in claim 1; C2) An expression cassette containing the nucleic acid molecule described in C1); C3) A recombinant vector containing the nucleic acid molecule described in C1), or a recombinant vector containing the expression cassette described in C2); C4) Recombinant microorganisms containing the nucleic acid molecules described in C1), or recombinant microorganisms containing the expression cassette described in C2), or recombinant microorganisms containing the recombinant vector described in C3).

3. The related biomaterial according to claim 2, characterized in that, The nucleic acid molecule is any one of the following: B1) The DNA molecule shown in SEQ ID No. 1; B2) A DNA molecule that hybridizes under stringent conditions with the DNA molecule defined in (B1) and encodes the protein thereon; B3) has a DNA sequence that is more than 75% identical to that of B1) and encodes the protein therein; B4) The DNA molecule shown in SEQ ID No. 2; B5) A DNA molecule that hybridizes under stringent conditions with the DNA molecule defined in (B4) and encodes the protein thereon; The DNA molecule that has more than 75% DNA sequence identity with B4) and encodes the protein described therein; B7) The DNA molecule shown in SEQ ID No. 3; B8) A DNA molecule that hybridizes under stringent conditions with the DNA molecule defined in (B7) and encodes the protein thereon; DNA molecules that have more than 75% DNA sequence identity with B7) and encode the protein described therein; The DNA molecule shown in SEQ ID No. 4 (B10); B11) A DNA molecule that hybridizes under stringent conditions with a DNA molecule defined by (B10) and encodes the protein thereon; The DNA sequence of B12) and B10) has more than 75% identity and encodes the DNA molecule of the protein.

4. The application of the protein of claim 1 or the related biomaterials of claim 2 or 3 in the synthesis of phenolamide derivatives.

5. The application according to claim 4, characterized in that, The phenolamide derivative acyltransferase catalyzes the synthesis of phenolamide derivatives from polyamines and hydroxycinnamoyl coenzyme A.

6. The application according to claim 5, characterized in that, The phenolamide derivative acyltransferases synthesize N,N'-dicaffeoyl spermidine, N,N'-dicumaroyl spermidine, N,N'-dicaffeoyl spermidine, N,N'-dicumaroyl spermidine, or N,N',N”-tricaffeoyl spermidine.

7. A method for preparing phenolamide derivatives, characterized in that, The step includes using the protein described in claim 1 as a phenolamide derivative acyltransferase to perform an enzymatic reaction.

8. The method for preparing phenolamide derivatives according to claim 7, comprising the step of using the protein according to claim 1 as an acyltransferase for phenolamide derivatives to catalyze the generation of N,N'-dicaffeoyl spermine, N,N'-dicoumaroyl spermine and N,N',N”-tricaffeoyl spermine from spermine.

9. The method for preparing phenolamide derivatives according to claim 7, comprising the step of using the protein according to claim 1 as an acyltransferase for phenolamide derivatives to catalyze the generation of N,N'-dicaffeoylspermine and N,N'-dicoumaroylspermine from spermidine.

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