CN109971808B

CN109971808B - Process for preparing malonylated products

Info

Publication number: CN109971808B
Application number: CN201711454456.5A
Authority: CN
Inventors: 屠鹏飞; 刘晓; 史社坡; 李军; 刘雨雨; 闫雅如; 王晓晖; 宋月林; 莫婷; 王娟
Original assignee: Beijing University of Chinese Medicine
Current assignee: Beijing University of Chinese Medicine
Priority date: 2017-12-28
Filing date: 2017-12-28
Publication date: 2020-09-18
Anticipated expiration: 2037-12-28
Also published as: CN109971808A

Abstract

A process for producing a malonyl product comprising reacting a reaction system comprising a plant-derived universal malonyl transferase, a glycoside compound acceptor and an acyl donor source under suitable conditions to obtain a malonyl product; wherein the positive substrate of the universal malonyl transferase comprises O-glycoside and C-glycoside with different structure types. The preparation method adopts plant-derived universal malonyl transferase, can utilize various glycoside compounds as substrates, and makes up the defects of chemical modification and single-substrate enzymatic modification by catalyzing malonyl reaction through an enzymatic method.

Description

Process for preparing malonylated products

Technical Field

The invention relates to a method for preparing a malonyl product, in particular to a method for preparing the malonyl product based on universal malonyl transferase from plant sources.

Background

Malonylation plays an important role in improving the stability, lipid solubility, bioavailability and the like of the compound. Chemical and enzymatic methods are currently the two main approaches to achieve malonyl modification of compounds. The chemical method for realizing malonyl modification has the advantages of high acylation degree, low cost and the like, but the reaction process is not easy to control, the reaction conditions are harsh, particularly the product specificity is poor, and the structure of the obtained product and the proportion of each component are difficult to determine. The enzymatic acylation has the advantages of strong specificity, high catalytic efficiency, mild reaction conditions, controllable process, environmental friendliness and the like, so that the enzymatic acylation is a powerful supplement of a chemical method and plays an increasingly important role in reactions in which chemical modification is difficult to realize.

As the catalytic enzyme in the enzymatic acylation, a plant-derived malonyl transferase can be used. Research on plant-derived malonyl transferases has progressed. For example, Suzuki H et al have cloned in soybean the malonyl transferase gene GmIF7MaT which catalyzes the malonyl-ation of the isoflavone 7-O-glucoside-6' -O-malonyl. Luo J and the like adopt an improved functional genomics research strategy, combine the co-expression profile and the accumulation condition of anthocyanin for analysis, and identify genes capable of coding 3 anthocyanin acyltransferases from arabidopsis thaliana. Yu XH et al identified 3 malonyl transferase genes MtMaT1, MtMaT2, and MtMaT3 from the legume Medicago truncatula, and the expression products of these three genes could specifically recognize malonyl coenzyme A as a receptor, catalyze the malonyl reaction of isoflavone 7-O-glucoside, and show specific tissue differential expression pattern and different stress response to biotic and abiotic stress. Taguchi G et al cloned and expressed a malonyl transferase NtMaT1 that catalyzes the malonyl-ation of various phenolic glycoside compounds such as flavone 7-O-glucoside, flavone 3-O-glucoside, and naphthol glycoside compounds in tobacco.

As described above, although studies on plant-derived malonyl transferases have been advanced, the enzymes identified so far are only directed to malonyl of a specific compound or a specific class of compounds, and there are no general reports on malonyl transferases. Therefore, when a foreign compound requires malonyl substitution, if the structural type of the foreign compound is significantly different from that of the malonyl transferase substrate, the foreign compound cannot be structurally modified by an enzyme-catalyzed method.

In addition, in the in vitro enzymatic malonyl reaction, in order to ensure the reaction to be fully carried out, excessive malonyl-coa is required as an acyl donor, which is expensive and easily degradable, thus limiting the application of enzymatic malonyl catalysis to some extent.

Disclosure of Invention

In order to solve at least part of the problems in the prior art, the inventor has obtained a universal malonyl transferase from plant source, which can accept glycoside compounds with various structural types as receptors, as a catalytic enzyme, so that the diacylation of glycoside compounds with different structural types can be realized. The present invention has been accomplished based on this. Specifically, the present invention includes the following.

A method of producing a malonyl product, comprising reacting a reaction system comprising a plant-derived universal malonyl transferase, a glycoside compound, and an acyl donor source under suitable conditions to obtain a malonyl product; wherein the positive substrate of the universal malonyl transferase comprises glycosidic compounds of different structural types, including both monoglycosides and polyglycosides, including both O-glycosides and C-glycosides.

In certain embodiments, the universal malonyl transferase is derived from cistanche tubulosa. Preferably, the universal malonyl transferase comprises a mutation and has malonyl catalytic activity, and the amino acid sequence thereof is shown in SEQ ID NO 1.

In certain embodiments, the glycoside compound is selected from at least one of the group consisting of phenylethanoid glycosides, stilbene glycosides, naphthol glycosides, anthraquinone glycosides, flavonoid glycosides, dihydroflavonoid glycosides, flavonol glycosides, isoflavone glycosides, flavone glycosides, coumarin glycosides, iridoid glycosides, lignan glycosides, unsaturated fatty acid glycosides, cyanogenic glycosides, terpene saponins, and dihydrochalcone glycosides.

In certain embodiments, the acyl donor source is malonyl-coa or a malonyl-donor synthesis system. Preferably, the malonyl donor synthesis system comprises malonic acid, coenzyme a, ATP, DTT, a divalent ion and a catalytic enzyme. More preferably, the catalytic enzyme is malonyl-coa synthetase.

In certain embodiments, the reaction system comprises 1-50 μ g/100 μ L of a universal malonyl transferase, 0.01-2 mM of a glycoside compound, and 0.01-2 mM of a malonyl donor source. Preferably, the reaction system further comprises a buffer.

In certain embodiments, suitable conditions include a reaction temperature of 20 to 40 ℃ and a reaction time of 4 to 20 hours.

The preparation method adopts plant-derived universal malonyl transferase, can utilize various glycoside compounds as substrates, and makes up the defects of chemical modification and single-substrate enzymatic modification by catalyzing malonyl reaction through an enzymatic method.

In certain embodiments, the preparation methods of the present invention also establish a "one-pot" reaction system for malonyl-coa synthetase and malonyl transferase. The system changes the directly input acyl donor into an acyl donor synthesis system with a corresponding amount, so that once the acyl donor is produced, the acyl donor can directly enter the next malonyl catalysis process, thereby effectively realizing the direct synthesis of compounds such as cheap and easily obtained malonic acid and the like to obtain corresponding malonyl products, simplifying the whole reaction step, and simultaneously avoiding the possible degradation of unstable acyl donor malonyl coenzyme A in the intermediate treatment process to the maximum extent.

Drawings

FIG. 1 agarose gel electrophoresis of the coding region of the gene for malonyl transferase CtMaT1 with the coding region of the gene for malonyl-CoA synthetase MatB.

FIG. 2 is an SDS-PAGE electrophoresis of malonyl transferase CtMaT1 obtained by heterologous expression. Wherein Line 1-2 is 70mM imidazole elution fraction, and Line 3-4 is 110mM imidazole elution fraction; line 5-6 is 160mM imidazole elution fraction; line7 was a 250mM imidazole elution fraction.

FIGS. 3 to 34 are HPLC-MS analysis charts of malonyl reaction of 32 positive substrates of different structure types catalyzed by malonyl transferase CtMaT 1.

FIG. 35 is a reaction scheme of malonyl transferase CtMaT1 catalyzing malonyl-ation of salidroside glucose 6-OH.

FIG. 36 is a reaction scheme in which malonyl transferase CtMaT1 catalyzes malonyl-conversion of icariin glucose 6-OH.

FIG. 37 is a schematic diagram of the structure of a positive glycoside substrate catalyzed by malonyl transferase CtMaT 1.

FIG. 38 is a graph comparing yields of malonyl products in 32 positive substrate single enzymatic reactions and in a "one-pot" reaction.

In FIGS. 3 to 34, the positive substrates are 1 to 32, respectively, and the malonyl products are 1a to 32a, respectively; in FIGS. 3 to 34, the large graph is an HPLC graph, and the small graph is a DAD ultraviolet characteristic absorption spectrum graph of the positive substrate and the malonyl product thereof, respectively. FIG. 37 shows the structural formulae of the 32 positive glycoside substrates (1 to 32) of FIGS. 3 to 34. The positive substrates 1-32 are as follows:

phenylethanoid glycosides: salidroside (1), 2-OH phenylethanol glucoside (2), phenylethanol nidoside (3);

stilbene glucoside: polygonin (2,3,5,4' -tetrahydroxystilbene 2-O-glucoside) (4), polydatin (pieecid) (5);

and (3) naphthol glycoside: 2-naphthyl- β -D-glucopyranoside (2-naphthyl- β -D-glucopyranoside) (6);

anthraquinone glycoside: emodin-8-beta-D-glucopyranoside (emodin 8-O-glucoside) (7);

and (3) flavonoid glycoside: comprises flavonoid glycosides such as swertisin (8) and vitexin (9); flavanonoglycosides, such as naringin (10), liquiritin (11); flavonol glycosides, such as icariin (12), hyperoside (13), quercetin-3-O-glucose-7-O-rhamnoside (14); isoflavone glycosides, such as sophoricoside (15), puerarin (16), glycitin (17), tectoridin (18), formononetin (19), calycosin (7-glucoside) (20); (iii) ketonic glycosides (xanthones), such as mangiferin (mangiferin) (21);

coumarin glycoside: aesculin (22), 4-methylumbelliferyl-beta-galactopyranoside (23), 4-methylumbelliferyl-beta-D-glucopyranoside (24);

lignan glycosides: seco-isolariciresinol diglucoside (25);

iridoid glycosides: gentiopicroside (26), geniposide (27);

unsaturated fatty acid glycosides: crocin (crocin) (28);

cyanogenic glycosides: amygdalin (amygdalin) (29);

terpenoid saponins: ginsenoside Rb1(30), saikosaponin a (31);

dihydrochalcone glycoside: phlorizin (32).

Detailed Description

The present invention will be further described with reference to the following specific examples, but the scope of the present invention is not limited thereto.

The invention clones an enzyme with malonyl group transfer function from the traditional Chinese medicine cistanche tubulosa for the first time, and obtains the universal malonyl transferase CtMaT1 with high activity and stability through further screening, identification and optimization. The present invention has been accomplished based on this.

The present invention provides a method for producing a malonyl product comprising reacting a reaction system comprising a plant-derived universal malonyl transferase, a glycoside compound, and an acyl donor source under suitable conditions to obtain a malonyl product. Herein, the "reaction system comprising a plant-derived universal malonyl transferase, a glycoside compound and an acyl donor source" is sometimes simply referred to as "reaction system".

The source of the malonyl transferase is plant cistanche tubulosa (Schenk) Wight). Various acyltransferases are present in cistanche tubulosa. The invention firstly screens and identifies the cistanche tubulosa to obtain universal malonyl transferase CtMaT 1. The malonyl transferase of the present invention can be a native protease or an enzyme that comprises a mutation and still has malonyl catalytic activity. Preferably, the malonyl transferase of the present invention comprises a mutation such that it has a stronger malonyl catalytic activity. Preferably, the malonyl transferase of the present invention comprises a mutation to confer other beneficial properties, such as greater stability or heat resistance, etc., while maintaining malonyl catalytic activity. More preferably, the malonyl transferase of the present invention is CtMaT1 transferase comprising the amino acid sequence shown in SEQ ID No. 1. In certain embodiments, the amino acid sequence of the malonyl transferase has greater than 90%, preferably greater than 92%, more preferably greater than 95%, even more preferably greater than 98%, and yet more preferably greater than 99% homology to the sequence set forth in SEQ ID NO. 1 and is derived from cistanche deserticola of the same species. "homology" in this application refers to the similarity between two sequences, which can be determined by any algorithm known in the art. For example, the degree of identity between two amino acid sequences can be determined using the Needleman-Wunsch algorithm. Preferably, the malonyl transferase ctmut 1 of the present invention has a molecular weight of 50.0KDa, a theoretical isoelectric point of 5.65, and consists of 448 amino acids.

The "glycoside compound" as referred to herein means a compound in which a sugar or a derivative of a sugar is glycosidically bonded to a ligand. Including both monoglycoside and polyglycoside compounds; glycoside compounds of both the O-glycoside and C-glycoside type; wherein the monoglycoside and polyglycoside are classified according to the number of sugar chain substitutions, and the O-glycoside and C-glycoside are classified according to the number of glycosidic bond atoms.

The term "universality" as used herein means that the positive substrate for malonyl transferase includes both mono-and polyglycosides and also O-and C-glycoside type compounds. That is, the universal malonyl transferase can catalyze the reaction of both a monoglycoside and an acyl donor to produce a malonyl product and a polyglycoside and an acyl donor to produce a malonyl product; in addition, it is possible to catalyze the reaction of an O-glycoside type compound with an acyl donor to produce a malonyl product and also a C-glycoside type compound with an acyl donor to produce a malonyl product, but glycoside compounds in which the glycosidic bond atom is an atom other than oxygen or carbon are not excluded.

Glycoside compounds of the O-glycoside type include, but are not limited to, alcohol glycosides, phenol glycosides, ester glycosides, cyanogenic glycosides, and the like. Examples of the alcohol glycosides include salidroside, phenylethanoid vicine, and the like. Examples of the phenolic glycoside include polygonin, polydatin, emodin-8-beta-D-glucopyranoside, and the like. Examples of the ester glycosides include crocin and the like. Examples of cyanogenic glycosides include, but are not limited to, amygdalin and the like. C-glycoside type glycoside compounds include mangiferin, vitexin, etc.

The sugar or sugar derivative moiety in the glycoside compound includes a monosaccharide or polysaccharide, such as disaccharide, trisaccharide and the like. The monosaccharides may include alpha-or beta-isomers. Examples of monosaccharides include, but are not limited to, glucose, galactose, rhamnose and xylose, and their derivatives. As the aglycone part in the glycoside compound, a non-sugar substance is preferable, and examples thereof include phenethyl alcohol, stilbene, naphthol, anthraquinone, flavone, coumarin, iridoid, lignan, unsaturated fatty acid and the like.

Specific examples of the glycoside compounds described in the present invention include, but are not limited to, phenylethanoid glycosides, stilbene glycosides, naphthol glycosides, anthraquinone glycosides, flavonoid glycosides, dihydroflavonoid glycosides, flavonol glycosides, isoflavone glycosides, flavone glycosides, coumarin glycosides, iridoid glycosides, lignan glycosides, unsaturated fatty acid glycosides, cyanogenic glycosides, terpene saponins, and dihydrochalcone glycosides.

Examples of the phenylethanoid glycoside compounds include salidroside, 2-OH phenylethanol glucoside, phenylethanoid glucosides, and the like; examples of the stilbene glycoside compound include polygonin, polydatin, and the like; examples of the naphthol glycoside compound include 2-naphthyl- β -D-glucopyranoside and the like; examples of the anthraquinone glycoside compound include emodin-8- β -D-glucopyranoside and the like; examples of the flavonoid glycoside compounds include swertisin, vitexin and the like; examples of the flavanone glycoside compound include naringin, liquiritin and the like; examples of the flavonol glycoside compounds include icariin, hyperoside, quercetin-3-O-glucose-7-O-rhamnoside, and the like; examples of the isoflavone glycoside compounds include sophoricoside, puerarin, glycitin, tectoridin, formononetin, calycosin, and the like; examples of the xanthone compound include mangiferin and the like; examples of the coumarin glycoside compound include aesculin, 4-methylumbelliferone- β -galactopyranoside, 4-methylumbelliferone- β -D-glucoside, and the like; examples of the iridoid glycoside compounds include gentiopicroside, gardenoside and the like; examples of the lignan glycoside compound include secoisolariciresinol diglucoside and the like; examples of the unsaturated fatty acid glycoside compounds include crocin and the like; examples of the cyanogenic glycoside compound include amygdalin and the like; examples of the terpenoid saponin compounds include ginsenoside Rb1, saikosaponin a, etc.; examples of the dihydrochalcone glycoside compound include phlorizin and the like. The present invention may use one or a combination of more of the above substances. Preferably, the present invention uses various glycoside compounds among the above-mentioned substances as a substrate.

The term "acyl donor source" as used herein refers to a compound or synthetic system capable of providing an active acyl donor for a reaction. Preferred as the compound is malonyl-coenzyme A. Any compound known so far can be used for malonyl-coa.

As the synthesis system, a synthesis system comprising malonic acid, coenzyme A, ATP, DTT, divalent ions and a catalytic enzyme including malonyl coenzyme A synthetase is preferable. Any type known so far can be used for malonyl-coa synthetase. For example, commercially available malonyl-coa synthetase, or malonyl-coa synthetase obtainable by biotechnological means, may be used. The source of malonyl-coa synthetase is not particularly limited. For example, it may be derived from Arabidopsis thaliana. In certain embodiments, malonyl-coa synthetase MatB is used, and its cloning and exogenous expression methods are described in the documents h.chen, h.u.kim, h.weng and j.browse, Plant Cell, 2011. In certain embodiments, the synthesis system further comprises a buffer. Preferably, the pH of the buffer is in the range of 6.8-7.2, preferably 7.0. The buffer may be a phosphate buffer, for example, a potassium phosphate buffer or a sodium phosphate buffer, etc. The concentration of the salt in the buffer is not particularly limited as long as the pH range according to the present invention can be provided. Preferably, the concentration of the salt substance is 50-150 mM, preferably 100 mM.

In the reaction system of the invention, the concentration of the universal malonyl transferase is between 1 and 50 mug/100 muL, preferably between 2 and 45 mug/100 muL, further preferably between 5 and 40 mug/100 muL, and further preferably between 10 and 30 mug/100 muL. The concentration of the glycoside compound is in the range of 0.01 to 2mM, preferably 0.1 to 1mM, and more preferably 0.2 to 0.8 mM. In the case where the malonyl donor source is malonyl-CoA, the concentration thereof is 0.01 to 2mM, preferably 0.1 to 1mM, and more preferably 0.2 to 0.8 mM; when the malonyl donor source is a synthesis system, the concentration or content of each component in the synthesis system is not particularly limited as long as it can provide malonyl-CoA at a concentration of 0.01 to 2mM, preferably in the range of 0.1 to 1mM, and more preferably in the range of 0.2 to 0.8 mM.

Preferably, the reaction system of the present invention further comprises a buffer. Preferably, the pH of the buffer is in the range of 6.8-7.2, preferably 7.0. Preferably, the buffer may be a phosphate buffer, for example, a potassium phosphate buffer or a sodium phosphate buffer, etc. The concentration of the salt in the buffer is not particularly limited as long as the pH range according to the present invention can be provided. Preferably, the concentration of the salt substance is 50-150 mM, preferably 100 mM.

Preferably, the reaction system of the present invention may further comprise other materials required for the reaction. For example, ATP, DTT and MgCl₂And the like.

In the present invention, preferably, the reaction system and/or the malonyl donor synthesis system is an in vitro reaction system. In certain embodiments, the reaction system of the present invention and the malonyl donor synthesis system are each present separately. In certain embodiments, the reaction system of the present invention and the malonyl donor synthesis system are the same system. In this case, the same buffer can be used for both.

The "suitable conditions" described in the present invention are conditions that ensure the reaction between the glycoside compound and the acyl donor source. The conditions include a reaction temperature of 20 to 40 ℃, preferably 25 to 35 ℃, for example 30 ℃ and a reaction time of 4 to 20 hours, preferably 5 to 15 hours, more preferably 10 hours.

Example 1 acquisition of the Gene encoding malonyl transferase CtMaT1

1. Extraction of total RNA of fresh cistanche tubulosa explant and preparation of RACE template

Selecting fresh and tender tissues on cistanche tubulosa plants, cleaning the surfaces with clear water, absorbing residual water with absorbent paper, and extracting total RNA by using an RNA extraction kit RNA plant kit (Omega) by adopting a liquid nitrogen quick-freezing grinding method. The total RNA was analyzed by gel electrophoresis, and the concentration was measured by Nanodrop, and 5 '-RACE-cDNA, 5' -RACE-cDNA and total cDNA were obtained by reverse transcription using SMARTer RACE 5 '/3' Kit (ClonTech), respectively.

Obtaining of 5' -RACE-cDNA: mixing 0.2-2.0 μ g RNA with 1.0 μ L5' -CDS-primer A, ddH₂O to 3.75. mu.L, PCR step1(72 ℃,3 min; 42 ℃, 2min) was performed, after the reaction was completed, the mixture was immediately taken out on ice, 1.0. mu.L of SMARTER II A oligo and 4.25. mu.L of Mix (containing 5 × First-strand buffer 2.0. mu.L, 20mMDTT 1.0. mu.L, 10mM dNTP 1.0. mu.L, RNase Inhibitor 0.25. mu.L) were added, the mixture was mixed and instantaneously separated, then 1.0. mu.L of SMARTscribes reverse transcriptase was added, PCR step2(42 ℃, 90 min; 72 ℃, 10min) was performed, and after the reaction was completed, the product was diluted with 100. mu.L of RNase Water.

3' -RACE-cDNA: mixing 0.2-2.0 μ g RNA with 1.0 μ L3' -CDS-primer A, ddH₂O was made up to 4.75. mu.L, PCR step1 was performed (72 ℃,3 min; 42 ℃, 2min), and after completion, the mixture was immediately taken out on ice, 4.25. mu.L of LMix (5 × First-strand buffer 2.0. mu.L, 20mM DTT 1.0. mu.L, 10mM dNTP 1.0. mu.L, RNaseINHIBITOR 0.25. mu.L) was added thereto, and after mixing and instantaneous dissociation, SMARTScript Reverse transcriptase 1.0. mu.L was added to perform PCR-step2(42 ℃, 90 min; 72 ℃, 10min), and after completion of the reaction, the product was diluted with 100. mu.L of RNase-free Water.

Obtaining RT-cDNA:taking 0.2-2.0. mu.g RNA, and 10. mu.M oligo dT₂₀2.0μL，ddH₂the amount of O was adjusted to 5.0. mu.L, PCR step1(72 ℃ C., 3min) was performed, 4.0. mu.L of Mix (5 XFirst-strand buffer 2.0. mu.L, 20mM DTT 1.0. mu.L, 10mM dNTP 1.0) was added to the reaction product, mixed well and instantaneously detached, SMARTScript reverse transcriptase 1.0. mu.L was added thereto, PCR step2(42 ℃ C., 90 min; 70 ℃ C., 15min) was performed, and after completion of the reaction, the product was diluted with 10. mu.LRNase Water.

Rapid amplification of cDNA Ends by RACE

Cloning of the 5 'and 3' terminal sequences of the target Gene is referred to SMARTer^TMthe RACE cDNA Amplification Kit (Clontech) is required to design a 5 '/3' -RACE specific primer of a CtMaT1 sequence on the basis of analysis of transcriptome data of cistanche tubulosa obtained in the early stage by taking 5 '/3' -RACE-cDNA (about 40ng) obtained by RACE reverse transcription as a template, and simultaneously combining a Kit universal primer UPM to amplify a corresponding 5 '/3' end sequence by using a high-fidelity DNA Amplification enzyme KOD-Plus-Neo DNA Polymerase, wherein the RACE Amplification reaction system comprises the 5 '/3' -E-cDNA: 40ng, the UPM (10 mu M):1.0 mu L, the 5 '/3' -RACE specific primer (10 mu M):0.5 mu L, 10 × KOD buffer:1.0 mu L, MgSO₄(25mM): 0.6. mu.L, dNTPs (2mM): 1.2. mu.L, KOD DNA Polymerase (1U/. mu.L): 0.2. mu.L. The PCR reaction program is: pre-denaturation at 94 ℃ for 2 min; then denaturation at 94 ℃ for 15s, initial annealing at 65 ℃ for 30s, reduction of 0.5 ℃ per cycle, elongation at 68 ℃ for 50s, for 30 cycles; then denaturation at 94 ℃ for 15s, annealing at 54 ℃, extension at 30s and 68 ℃ for 50s for 25 cycles; finally, extension is carried out for 1min at 72 ℃.

The 5' end RACE specific primer sequence is shown as SEQ ID NO. 4; 5' end RACE PCR product 1300 bp.

The 3' end RACE specific primer sequence is shown as SEQ ID NO. 5; the RACE PCR product at the 3' end is 1400 bp.

The 5 'RACE and 3' RACE sequences obtained by sequencing are spliced to obtain a full-length cDNA sequence shown as SEQ ID NO. 3. Analyzing the cDNA fragment sequences obtained by 5 '-RACE and 3' -RACE, and designing a pair of specific primers with enzyme cutting sites, wherein the sequences are shown as SEQ ID NO. 6 and 7, the SEQ ID NO. 6 is introduced into EcoR I enzyme cutting sites through primer design, and the SEQ ID NO. 7 is introduced into Not I enzyme cutting sites through primer design. The full length of the coding region sequence of about 1347bp is obtained by amplification with a pair of specific primers, the sequence is shown as SEQ ID NO. 2, the coding amino acid sequence is shown as SEQ ID NO. 1, and the agarose gel electrophoresis is shown as figure 1.

Example 2 construction of prokaryotic expression vector of the CtMaT1 Gene of malonyl transferase and prokaryotic expression

The full-length gel containing the gene with the enzyme cutting site obtained by amplification in the example 1 is recovered, enzyme cutting is carried out at 37 ℃ for 2h, then the recovered product of the enzyme cutting gel and pET-28a are connected at 25 ℃ for 30min under the action of T4DNA Ligase of NEB, then the obtained product is immediately transformed into competent cells, and after culture at 37 ℃ for 12h, colony PCR reaction is carried out to screen positive clones and sequencing is carried out. Coli BL21(DE3) was selected as the expression strain. Inoculating the strain with correct sequencing into LB liquid medium containing Kana 50. mu.g/mL and Chl 50. mu.g/mL, and shake-culturing at 37 deg.C and 200rpm on a constant temperature shaker to OD₆₀₀And adding an inducer IPTG (isopropyl-beta-thiogalactoside) to the concentration of 0.5mM at 0.4-0.6, and culturing at 23 ℃ and 180rpm for 16h to induce the expression of the target protein at low temperature. According to the fact that the recombinant protein CtMaT1 contains a His tag at the N-terminal, CtMaT1 protein was purified by using a nickel ion affinity chromatography column (Ni Sepharose 6Fast Flow, GEHealthcare). The protein expression and purification results are shown in fig. 2, and the imidazole gradient elution obtains a single band of target protein. The fractions of the target protein were pooled and concentrated in PD-10 column. This was done using Easy protein quantitative Kit.

Example 3 in vitro enzyme Activity assay of the protein CtMaT1 malonyl transferase and HPLC-HR-MS detection of the reaction

CtMaT1 is prepared by in vitro enzymatic catalysis reaction system (150 μ L) including 0.4mM malonyl donor, 0.4mM malonyl acceptor, 10 μ g protease, potassium phosphate buffer (pH7.0, 100mM) to 150 μ L, mixing with light bomb, reacting at 30 deg.C for 12h, adding two volumes of chromatographic methanol to stop the reaction, and performing HPLC-HR-ESI-MS analysis under conditions of Agilent1260HPLC with a sample volume of 30 μ L under conditions of chromatographic COLUMN SHESHIHDO C18COLUMN (4.6mml.D. × 250mM, 5 μm), DAD detector full-wavelength scanning mobile phase containing A (0.5 ‰ formic acid water), and B (acetonitrile) eluting with a flow rate gradient of 1.0mL/min, and the procedure is described in the following A-B (v/v)From 0min (95:5) to 20min (40:60) to 28min (10:90) to 29min (2:98) to 35min (2: 98). And performing high-resolution mass spectrometry by using an Shimadzu LCMS-IT-TOF ion trap time-of-flight mass spectrometer. Wherein, the chromatographic column and the liquid phase conditions are the same as above, and the mobile phase is changed into the corresponding mass spectrum solvent. The mass spectrum condition parameters are respectively set as: positive and negative ion mode, automatic multi-stage MS¹、MS²、MS³Full scan, atomizing gas (nebulizing gas) N₂The flow rate is 1.5 ml/min; dry gas N₂pressure 100MPa, cooling gas and collision gas of collision-induced ionization (CID), interface voltage and detector voltage are both 1.40KV, vacuum degree of ion trap (IT vacuum, 1.9 × 10)^-2Pa); the heater temperature and the CDL temperature are both 200 ℃; the ion accumulation time is 100 ms; the CID collision energy was set to 50%, and data processing was performed using LC solvationversion 1.1 software (Shimadzu). And determining the generation of malonylation products by high performance liquid ultraviolet characteristic absorption spectrum and high resolution mass spectrum molecular weight analysis and molecular formula prediction.

The HPLC spectra and DAD ultraviolet characteristic absorption spectra of 32 positive substrates (1-32) and malonyl products (1 a-32 a) are shown in FIGS. 3-34; the high resolution mass spectral data are shown in table 1.

TABLE 1

Example 4 cloning and expression of malonyl-CoA synthetase MatB and construction of a "one-pot" reaction System for MatB and malonyl transferase CtMaT1

Cloning and exogenous expression of malonyl-coa synthetase MatB were performed according to the methods described in the literature (h.chen, h.u.kim, h.wengand j.browse, Plant Cell, 2011). A reaction system of synthesizing malonyl coenzyme A by utilizing MatB is used for preparing a 'one-pot method' reaction system. The details are as follows.

Reaction system for synthesizing malonyl coenzyme A by utilizing MatB150 μ L containing 1.5mM coenzyme A, 3mM malonic acid, 3mM MATP, 1mM DTT, 5mM MgCl ₂15. mu.g of MatB recombinant protein. After 12h reaction at 30 ℃ the reaction was stopped by adding 4% (w/v) ammonium acetate. The following "one-pot" reaction system was prepared with reference to this system.

The amount of acyl acceptor and protease CtMaT1 in the MatB and malonyl transferase CtMaT1 reaction system is unchanged compared with that in a single enzyme system, and the single enzyme system contains 0.4mM malonyl acceptor, 10 μ g CtMaT1, 1.5mM coenzyme A, 3mM malonic acid, 3mM ATP, 1mM DTT, and 5mM MgCl₂Mu.g of MatB, 15. mu.g of potassium phosphate buffer (pH7.0, 100mM) to 150. mu.L, and reacted in a water bath shaker at 30 ℃ for 5 hours at a rotation speed of 60 rpm. The reaction was stopped by adding twice the volume of chromatographic methanol, HPLC-HR-MS detection conditions were the same as in example 3, and malonylation product formation was determined by HPLC-UV characteristic absorption spectroscopy combined with high resolution mass spectrometry molecular weight analysis and molecular formula prediction.

HPLC-UV detection analysis shows that the 'one-pot' reaction system can realize all positive reactions in CtMaT1 single-enzyme reaction, and the same malonyl product is generated in the 'one-pot' reaction system; second, the receptor conversion was substantially equivalent in both methods compared to each other, and the conversion of some of the substrate in the "one-pot" reaction system was far beyond that of CtMaT1 single-enzyme reaction system, as shown in fig. 38. In addition, it is more worth mentioning that malonyl coenzyme A is synthesized by cheap and easily available substrates such as malonic acid, coenzyme A and the like through MatB enzymatic reaction and is directly used in the next malonyl reaction, so that the method is efficient, time-saving, quick and cost-saving; in addition, the generated malonyl coenzyme A is directly put into the subsequent acyl transfer reaction, thereby avoiding the degradation of the malonyl coenzyme A which is unstable chemically to the maximum extent, improving the reaction efficiency and having extremely high application value.

Example 5 application of malonyl transferase CtMaT1 to catalyze preparation and structural identification of malonyl products of salidroside and icariin

Malonylation amplification reaction of salidroside: 0.036mmol Salidroside (10.8mg), 0.135mmol coenzyme A, 0.27mmol malonic acid, 0.27mmol ATP, 6mg CtMaT1 and9mg of MatB recombinant protein in 90mL of reaction buffer (pH6.0, 100mM KPB, 1mM DTT, 5mM MgCl)₂) Reacting at 30 ℃ for 5h, adding 2 times volume of chromatographic methanol, shaking, mixing uniformly, filtering with a 0.45-micron filter membrane, performing HPLC detection on a small amount of filtrate to confirm the output condition of the product, performing reduced pressure concentration on the rest of filtrate by an R-210 rotary evaporator, re-dissolving the obtained residue with 5mL of chromatographic methanol, filtering the solution, and performing semi-preparative liquid phase separation and purification.

Malonylation amplification reaction of icariin: 0.0148mmol icariin (10.0mg), 0.0555mmol coenzyme A, 0.11mmol malonic acid, 0.11mmol ATP, 2.47mg CtAT5 and 3.7mg MatB recombinant protein in 37ml reaction buffer (pH6.0, 100mM KPB, 1mM DTT, 5mM MgCl)₂) Reacting at 30 ℃ for 5h, adding 2 times of volume of chromatographic methanol, shaking and uniformly mixing, filtering the reaction solution through a 0.45-micron filter membrane, taking out a small amount of filtrate, performing HPLC detection to confirm the output condition of the product, performing reduced pressure concentration on the rest of filtrate through an R-210 rotary evaporator, re-dissolving the residue with 2mL of chromatographic methanol, filtering the solution, and performing semi-preparative liquid phase separation and purification.

separating and purifying the reaction solution by Shimadzu semi-preparative liquid phase, wherein the preparative column is YMC-Pack ODS-A HPLCCOLUMN (10mml.D. × 250mm, 12 μm), the semi-preparative liquid phase elution procedure of salidroside malonyl product comprises mobile phase A (0.1% formic acid aqueous solution) and B (acetonitrile), the gradient elution procedure is 0min (98:2) to 5min (90:10) to 20min (60:40) to 25min (20:80) to 30min (0:100), the semi-preparative liquid phase elution procedure of icariin malonyl product comprises 0min (85:15) to 5min (60:40) to 15min (20:80) to 20min (0:100), the flow rate is 3mL/min, the effluent chromatographic peaks are respectively collected and combined according to different peak-emergence ×, and the specific peak-emergence time of the chromatographic peak is confirmed by Agilent HPLC detection, the prepared product stream is concentrated by an R-210 rotary evaporator, and then placed in A dry solvent box, and the appropriate amount of DZF is dissolved in Methanol for 90-60d substitution and dried₄In (1), the product is collected by an Innova-500 nuclear magnetic resonance apparatus¹H-NMR、¹³C-NMR, HSQC and HMBC spectra, combined with the software MestReNova, for structural identification of the product. The result of structural identification is shownIt is shown that the malonyl substitution of both compounds occurs at the 6-OH group of the glucose chain.

The present invention is not limited to the above-described embodiments, and any variations, modifications, and substitutions which may occur to those skilled in the art may be made without departing from the spirit of the invention.

SEQUENCE LISTING

<110> Beijing university of traditional Chinese medicine

<120> Process for producing malonylated product

<130>YC12017110028-A

<160>7

<170>PatentIn version 3.3

<210>1

<211>448

<212>PRT

<213>Cistanche tubulosa

<400>1

Met Thr Thr Thr Leu Leu Glu Thr Cys Arg Val Pro Pro Pro Ala Gly

1 5 10 15

Ala Ala Ala Val Leu Ser Val Pro Leu Ser Phe Phe Asp Phe Ile Trp

20 25 30

Ile His Phe His Pro Ile Arg Arg Leu Leu Phe Tyr Ser Tyr Pro Asn

35 40 45

Cys Ser Arg Pro Tyr Phe Leu Glu Thr Leu Ala Pro Gln Leu Lys Gln

50 55 60

Ser Leu Ser Leu Ala Leu Lys His Tyr Leu Pro Leu Ser Gly Asn Leu

65 70 75 80

Leu Tyr Pro Ser Asn Thr Glu Gln Lys Pro Val Phe Arg Tyr Val Asp

85 90 95

Gly Asp Ser Val Ser Leu Thr Val Ala Glu Ser Val Arg Asp Phe Asp

100 105 110

Glu Leu Val Gly Asn His Ala Arg Ser Ala Asp Gln Phe Tyr Asp Phe

115 120 125

Val Pro Glu Met Pro Gln Val Lys Asp Glu Pro Glu Tyr Lys Ile Val

130 135 140

Pro Val Leu Ala Leu Gln Val Thr Leu Phe Pro Asp Arg Gly Ile Cys

145 150 155 160

Ile Gly Phe Ala Asn His His Val Val Gly Asp Ala Ser Ser Ile Phe

165 170 175

Ser Phe Met Lys Thr Trp Ser Ser Ile Cys Ala Ala Glu Gln Ser Asp

180 185 190

His Pro Leu Pro Val Phe Asp Arg Ser His Ile Lys Asp Pro Leu Gly

195 200 205

Ile Asp Thr Ile Phe Trp Lys Val Met Arg Thr Ile Pro Phe Lys Pro

210 215 220

Ser Pro Phe Pro Leu Pro Thr Asn Arg Val Arg Ala Thr Phe Thr Leu

225 230 235 240

Arg Pro Ala Asp Ile Lys Lys Leu Lys Asp Leu Val Leu Ala Trp Lys

245 250 255

Pro Gly Leu Val Gln Val Ser Ser Phe Val Val Thr Ala Ser Tyr Val

260 265 270

Trp Thr Cys Leu Val Arg Ser Gly Asp Glu Ile Gly Glu Glu Val Asp

275 280 285

Gly Asp Val Pro Glu His Phe Ile Phe Val Val Asp Val Arg Gly Arg

290 295 300

Val Asp Pro Pro Val Pro Gly Asn Tyr Phe Gly Asn Cys Leu Gly Tyr

305 310 315 320

Val Leu Glu Lys Leu Glu His Lys Arg Val Val Gly Asp Asp Gly Phe

325 330 335

Val Ile Ala Ala Glu Gly Val Ala Glu Asp Ile Lys Lys Arg Val Asn

340 345 350

Asp Lys Asp Glu Val Leu Arg Gly Ala Glu Asn Trp Leu Ser Gly Phe

355 360 365

Glu Gly Tyr Gly Gly Met Arg Ala Met Gly Val Ser Gly Ser Pro Arg

370 375 380

Phe Asp Leu Tyr Gly Val Asp Phe Gly Trp Gly Arg Ala Arg Lys Leu

385 390 395 400

Glu Val Val Ser Ile Asp Gly Glu Ser Tyr Ser Met Ser Leu Cys Arg

405 410 415

Ser Asn Asp Ser Asp Gly Gly Leu Glu Ile Gly Leu Ser Leu Pro Lys

420 425 430

Lys Arg Met Glu Ala Phe Ala Ala Leu Phe Ala Glu Gly Leu Arg Phe

435 440 445

<210>2

<211>1347

<212>DNA

<213>Cistanche tubulosa

<400>2

atgactacca cactgcttga aacatgtcgc gtcccacctc cggccggcgc cgccgccgtg 60

ctatcggtcc ctctttcttt tttcgatttt atctggatcc acttccatcc catccgccgc 120

cttctcttct acagctatcc taactgttcc aggccctatt tcctggaaac cctcgccccg 180

caactcaaac aatcactttc cctcgctcta aaacactacc tccccttatc aggcaatttg 240

ctctaccctt caaacaccga gcaaaagcct gttttccgtt acgttgacgg cgactctgtc 300

tcgcttacgg ttgccgagtc cgtgcgtgat ttcgacgagc tcgtcggaaa ccatgcccgc 360

tctgctgatc agttttacga ttttgtcccc gaaatgccac aagtaaaaga cgaacccgaa 420

tacaaaatag tccccgtttt agcactgcag gtgacccttt tccctgatcg cggcatatgt 480

atcggtttcg caaatcacca cgttgtcggc gacgcgagct ccattttcag cttcatgaag 540

acatggtctt caatctgcgc cgcggaacaa tctgatcacc ctctaccggt tttcgacagg 600

tcccatatta aggatccgct cggaatcgac accatattct ggaaagtaat gagaacaata 660

ccattcaagc cgtcgccttt cccgttaccc acaaacaggg tccgggcaac atttactctc 720

cgcccggccg atataaagaa gctcaaggac ctggttctgg cctggaaacc gggtctggtt 780

caggtctcgt ctttcgtcgt cacggcgtct tacgtctgga cctgtttggt aagatccgga 840

gacgagattg gtgaggaggt ggacggggac gtgccggaac acttcatctt cgtggtcgac 900

gttaggggac gggtagatcc acccgttccc gggaattact tcggcaactg cttaggctac 960

gtgttggaga agttggagca taaacgggtg gtgggggatg acgggtttgt gattgctgcg 1020

gagggtgtcg cggaggacat caagaagagg gtgaacgata aggatgaagt gttgaggggt 1080

gctgagaatt ggttgtcggg tttcgagggt tacgggggga tgagggcaat gggggtgtcc 1140

ggttcgccgc ggttcgactt gtacggtgtg gatttcgggt ggggaagggc gaggaagctg 1200

gaggtcgtgt ccattgatgg agagagttat tcgatgtcgc tgtgtaggtc taatgattca 1260

gatggagggt tggagattgg gctgtctctt ccaaagaaaa ggatggaggc ttttgctgct 1320

ttatttgctg agggattgag gttttga 1347

<210>3

<211>1867

<212>DNA

<213>Cistanche tubulosa

<400>3

ctaatacgac tcactatagg gcaagcagtg gtatcaacgc agagtacatg gggacatgac 60

tttgtctaat ttcctaatac tattttaatt taatctgatc tccagtagag taggtattga 120

aattggaaat tctccttttt taatctgatc gtccacaatt caattaaaat ttcggtagat 180

cgtccacaag attttgccta attccccaat attttaattt aatgtaatct cctcctccgc 240

ccttgactcc ccaataaata gctaatttct tcttaaaaga agcgcctggc atgactacca 300

cactgcttga aacatgtcgc gtcccacctc cggccggcgc cgccgccgtg ctatcggtcc 360

ctctttcttt tttcgatttt atctggatcc acttccatcc catccgccgc cttctcttct 420

acagctatcc taactgttcc aggccctatt tcctggaaac cctcgccccg caactcaaac 480

aatcactttc cctcgctcta aaacactacc tccccttatc aggcaatttg ctctaccctt 540

caaacaccga gcaaaagcct gttttccgtt acgttgacgg cgactctgtc tcgcttacgg 600

ttgccgagtc cgtgcgtgat ttcgacgagc tcgtcggaaa ccatgcccgc tctgctgatc 660

agttttacga ttttgtcccc gaaatgccac aagtaaaaga cgaacccgaa tacaaaatag 720

tccccgtttt agcactgcag gtgacccttt tccctgatcg cggcatatgt atcggtttcg 780

caaatcacca cgttgtcggc gacgcgagct ccattttcag cttcatgaag acatggtctt 840

caatctgcgc cgcggaacaa tctgatcacc ctctaccggt tttcgacagg tcccatatta 900

aggatccgct cggaatcgac accatattct ggaaagtaat gagaacaata ccattcaagc 960

cgtcgccttt cccgttaccc acaaacaggg tccgggcaac atttactctc cgcccggccg 1020

atataaagaa gctcaaggac ctggttctgg cctggaaacc gggtctggtt caggtctcgt 1080

ctttcgtcgt cacggcgtct tacgtctgga cctgtttggt aagatccgga gacgagattg 1140

gtgaggaggt ggacggggac gtgccggaac acttcatctt cgtggtcgac gttaggggac 1200

gggtagatcc acccgttccc gggaattact tcggcaactg cttaggctac gtgttggaga 1260

agttggagca taaacgggtg gtgggggatg acgggtttgt gattgctgcg gagggtgtcg 1320

cggaggacat caagaagagg gtgaacgata aggatgaagt gttgaggggt gctgagaatt 1380

ggttgtcggg tttcgagggt tacgggggga tgagggcaat gggggtgtcc ggttcgccgc 1440

ggttcgactt gtacggtgtg gatttcgggt ggggaagggc gaggaagctg gaggtcgtgt 1500

ccattgatgg agagagttat tcgatgtcgc tgtgtaggtc taatgattca gatggagggt 1560

tggagattgg gctgtctctt ccaaagaaaa ggatggaggc ttttgctgct ttatttgctg 1620

agggattgag gttttgagtt ttgtattggt cctcttatga tttttctttt tttaaccatt 1680

catctaacgt caagacatgt cacgagatac ataccacgat cggcgaaata tgtcgagcgt 1740

taacatgttt ttgtattcgt accaggattg aattttttat tggaagacga tgtcttgttt 1800

cttatttcat tgtatttgat gattatcatg ttttccaaaa aaaaaaaaaa aaaaaaaaaa 1860

aaaaaaa 1867

<210>4

<211>26

<212>DNA

<213> Artificial sequence

<400>4

tccgcagcaa tcacaaaccc gtcatc26

<210>5

<211>26

<212>DNA

<213> Artificial sequence

<400>5

tcgccccgca actcaaacaa tcactt 26

<210>6

<211>28

<212>DNA

<213> Artificial sequence

<400>6

ccggaattca tgactaccac actgcttg 28

<210>7

<211>28

<212>DNA

<213> Artificial sequence

<400>7

gcggccgcaa acctcaatcc ctcagcaa 28

Claims

1. A method for producing a malonyl product, comprising reacting a reaction system comprising a plant-derived universal malonyl transferase, a glycoside compound as an acceptor, and an acyl donor source under suitable conditions to obtain a malonyl product;

wherein the universal malonyl transferase is CtMaT1 transferase derived from cistanche tubulosa, and the amino acid sequence of the universal malonyl transferase is shown as SEQ ID NO. 1;

the glycoside compound is selected from salidroside, 2-OH phenylethanol glucoside, phenylethanol nidoside, polygonin, polydatin, 2-naphthyl-beta-D-glucopyranoside, emodin-8-beta-D-glucopyranoside, swertisin, vitexin, naringin, liquiritin, icariin, hyperoside, quercetin-3-O-glucose-7-O-rhamnoside, sophoricoside, puerarin, glycitin, tectoridin, formononetin, calycoside, mangiferin, aesculin, 4-methylumbelliferone-beta-galactopyranoside, 4-methylumbelliferone-beta-D-glucoside, secoisolaricoside, gentiopicroside, crocin, crocetin, naringin, 4-methylumbelliferone-beta-D-glucoside, secoisolaricoside, geniposide, and the like, Amygdalin, ginsenoside Rb1, saikosaponin A or phlorizin.

2. The method of claim 1, wherein the acyl donor source is malonyl-coa or a malonyl-donor synthesis system.

3. The method of claim 2, wherein the malonyl donor synthesis system comprises malonate, coenzyme a, ATP, DTT, divalent ions, and a catalytic enzyme.

4. The method of claim 3, wherein the catalytic enzyme is malonyl-CoA synthetase.

5. The method of claim 1, wherein the reaction system comprises 1-50 μ g/100 μ L of the universal malonyl transferase, 0.01-2 mM of the glycoside compound, and 0.01-2 mM of a malonyl donor source.

6. The method of claim 5, wherein the reaction system further comprises a buffer.

7. The method according to claim 1, wherein the suitable conditions include a reaction temperature of 20 to 40 ℃ and a reaction time of 4 to 20 hours.