Epoxide amide synthetase, method for synthesizing epoxide amide and application of epoxide amide synthetase

Abstract

The invention provides epoxide amide synthetase, a method for synthesizing epoxide amide and application of the epoxide amide synthetase. The invention discloses a class of epoxide amide synthetase for the first time, and establishes a method for synthesizing epoxide amide by catalyzing strictosamide by utilizing the epoxide amide synthetase. According to the invention, biological synthesis of epoxide amide is realized for the first time, and an effective way is provided for producing alkaloids such as camptothecin based on strategies such as synthetic biology.

Description

Technical Field

[0001] The present invention belongs to the field of enzymology and synthesis of active compounds, and more specifically, the present invention relates to epoxide amide synthase, a method for synthesizing epoxide amide and application thereof. Background Art

[0002] Camptothecin (CPT) is an alkaloid isolated from Camptotheca acuminate Decne, a plant of the Davidiaceae family endemic to China. It can inhibit DNA topoisomerase (TOPOI) and is one of the most studied natural anti-tumor drugs.

[0003] Strictosamide epoxide belongs to the monoterpene indole alkaloids. In the isotope test, by feeding the substrate deuterated labeled tryptamine to Camptotheca acuminata, deuterated labeled vinca glycoside lactam (Strictosamid), deuterated labeled epoxide amide and deuterated labeled camptothecin can be detected, which also proves that epoxide amide is an intermediate in the biosynthesis of camptothecin. Figure 1 , isovinca lactam is first converted into epoxide amide, and then the epoxide amide can be converted into camptothecin through a series of redox steps.

[0004] However, in the art, the synthesis route of epoxide amide is still unclear, and there is no report on the synthesis of epoxide amide using biological or chemical methods. Summary of the invention

[0005] The purpose of the present invention is to provide epoxide amide synthetase, a method for synthesizing epoxide amide and application.

[0006] In a first aspect of the present invention, a method for synthesizing epoxide amide is provided, comprising: catalyzing isovinca glycoside lactam with epoxide amide synthase to synthesize the epoxide amide; wherein the epoxide amide synthase is a polypeptide having an amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:10 or a conservative sequence variant thereof.

[0007] In one or more embodiments, the epoxide amide synthase is an enzyme derived from Snakeroot davidiana.

[0008] In one or more embodiments, the method is performed intracellularly; preferably, an expression cassette of epoxide amide synthetase is introduced into the cell to express epoxide amide synthetase, thereby catalyzing the isovinca glycoside lactam and synthesizing the epoxide amide.

[0009] In one or more embodiments, vinblastine is added to the cells as a substrate.

[0010] In one or more embodiments, the cell is a cell capable of expressing epoxide amide synthetase.

[0011] In one or more embodiments, the cells include: tobacco cells.

[0012] In another aspect of the present invention, a use of an epoxide amide synthetase is provided for catalyzing iso-catharanthus glycoside lactam and synthesizing the epoxide amide, wherein the epoxide amide synthetase is a polypeptide having an amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:10 or a conservative sequence variant thereof.

[0013] In one or more embodiments, the catalysis is performed within a cell.

[0014] In one or more embodiments, an epoxide amide synthetase expression cassette is introduced into the cell to catalyze the synthesis of the epoxide amide from isovinblastine lactam.

[0015] In one or more embodiments, the expression cassette is contained in a recombinant expression vector.

[0016] In another aspect of the present invention, an isolated epoxide amide synthetase is provided, which is a polypeptide having an amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10 or a conservative sequence variant thereof.

[0017] In one or more embodiments, the conservative variant polypeptide comprises:

[0018] (1) A polypeptide having the function of synthesizing epoxide amide formed by substitution, deletion or addition of one or more (e.g., 1-20, preferably 1-10; more preferably 1-5; more preferably 1-3) amino acid residues of the polypeptide of the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:10; and having the function of synthesizing epoxide amide;

[0019] (2) a polypeptide having an amino acid sequence that is 80% (preferably 85% or more; more preferably 90% or more; more preferably 95% or more; more preferably 99% or more) identical to the polypeptide shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10, and having the function of synthesizing epoxide amide; or

[0020] (3) A polypeptide formed by adding a tag sequence to the N-terminus or C-terminus of a polypeptide having an amino acid sequence as shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10, or by adding a signal peptide sequence to its N-terminus and retaining its function.

[0021] In another aspect of the present invention, an isolated polynucleotide is provided, which comprises a nucleotide sequence selected from the group consisting of:

[0022] (1) a polynucleotide encoding the epoxide amide synthetase as described above;

[0023] (2) A polynucleotide complementary to the polynucleotide (1).

[0024] In one or more embodiments, the polypeptide corresponding to the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:10, the nucleotide sequence of the polynucleotide is shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:9.

[0025] In another aspect of the present invention, an expression construct (such as an expression vector) is provided, which contains the polynucleotide.

[0026] In another aspect of the present invention, a recombinant cell is provided, which includes an exogenously introduced epoxide amide synthase expression cassette (such as the expression construct); the epoxide amide synthase is a polypeptide having an amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10 or a conservative sequence variant thereof.

[0027] In another aspect of the present invention, a method for preparing the recombinant cell is provided, comprising: introducing an epoxide amide synthase expression cassette into a cell to obtain the recombinant cell; the epoxide amide synthase is a polypeptide having an amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:10 or a conservative sequence variant thereof; preferably, the expression cassette is contained in a recombinant expression vector.

[0028] In one or more embodiments, the cell comprises: a prokaryotic cell or a eukaryotic cell.

[0029] In one or more embodiments, the prokaryotic cell comprises Escherichia coli or Streptomyces.

[0030] In one or more embodiments, the eukaryotic cells include plant cells, fungal cells, insect cells, and mammalian cells.

[0031] In one or more embodiments, the plant cell comprises: a tobacco cell.

[0032] In one or more embodiments, the eukaryotic cell comprises yeast.

[0033] In one or more embodiments, the yeast includes: Saccharomyces cerevisiae, Pichia pastoris, Schizosaccharomyces pombe, Kluyveromyces, and Candida.

[0034] In one or more embodiments, the cells include: algal cells.

[0035] In one or more embodiments, the cells include: cyanobacterial cells.

[0036] In one or more embodiments, the cells include: actinomycete cells.

[0037] In another aspect of the present invention, a kit for biosynthesis of epoxide amide is provided, comprising: the epoxide amide synthetase or its expression cassette; or the recombinant cell.

[0038] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 , schematic diagram of the biosynthesis of camptothecin.

[0040] Figure 2 , epoxide amide synthase catalytic results.

[0041] Figure 3, mass spectrometry results of products generated by snakeroot epoxide amide synthase. DETAILED DESCRIPTION

[0042] The inventors have devoted themselves to the study of the synthesis pathway of epoxide amides, and for the first time revealed a class of epoxide amide synthases, and established a method for producing epoxide amides. The technical solution of the present invention realizes the biological synthesis of epoxide amides, and provides an effective way for the production of alkaloids such as camptothecin based on strategies such as synthetic biology in the future.

[0043] Epoxide amide is an important precursor substance for the biosynthesis of camptothecin. It is generally believed that strictosamide is converted into epoxide amide under the catalysis of oxidase, but the enzyme that catalyzes the synthesis of epoxide amide from strictosamide has not been found, and there is no report on the synthesis of epoxide amide using chemical methods. The present invention provides a method for synthesizing epoxide amide by catalyzing epoxide amide synthase using strictosamide as a substrate.

[0044] Meanwhile, the epoxide amide synthetase of the present invention has the function of catalyzing the synthesis of epoxide amide from iso-catharanthus glycoside lactam, and can obtain epoxide amide through enzyme catalysis in different environments or multiple hosts (Escherichia coli, cyanobacteria, actinomycetes, yeast).

[0045] the term

[0046] As used herein, the term "epoxide amide" is used interchangeably with "Strictosamide epoxide".

[0047] As used herein, "exogenous" or "heterologous" refers to the relationship between two or more nucleic acid or protein sequences that are derived from different sources. For example, a promoter is exogenous to a gene of interest if the combination of the promoter and the gene of interest sequence is not normally found in nature. A particular sequence is "exogenous" to the cell or organism into which it is inserted.

[0048] As used in the present invention, the "expression cassette" refers to a gene expression system that contains all the necessary elements required to express the target gene, which generally includes the following elements: a promoter, a target gene sequence, a terminator, and optionally a signal peptide coding sequence, etc. These elements are operably connected to form the expression cassette.

[0049] As used in the present invention, the "element" refers to a series of functional nucleic acid sequences useful for the expression of proteins. In the present invention, the "element" is systematically constructed to form a nucleic acid construct. The sequence of the "element" can be those provided in the present invention, and also includes their variants, as long as these variants substantially retain the function of the "element".

[0050] As used in the present invention, the term "operably linked" or "operably connected" refers to the functional spatial arrangement of two or more nucleic acid regions or nucleic acid sequences. For example, a promoter is placed at a specific position relative to the target gene nucleic acid sequence, so that the transcription of the nucleic acid sequence is guided by the promoter, and thus the promoter is "operably linked" to the nucleic acid sequence.

[0051] Epoxide amide synthase

[0052] The inventors have discovered a novel epoxide amide synthetase by exploring plant active genes and combining in-depth research and experimental work. The epoxide amide synthetase of the present invention can be used to synthesize epoxide amide from isocatharanthus glycoside lactam in one step.

[0053] In a specific embodiment of the present invention, the epoxide amide synthetase has an amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:10.

[0054] The present invention also includes conservative variant polypeptides of the epoxide amide synthetase. In the present invention, the "conservative variant polypeptide" refers to a polypeptide that substantially maintains the same biological function or activity as the polypeptide. The "conservative variant polypeptide" may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substitution group in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a mature polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) a polypeptide formed by fusion of an additional amino acid sequence to this polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed with an antigen IgG fragment). According to the teachings of this article, these fragments, derivatives and analogs belong to the scope known to those skilled in the art.

[0055] The "conservative variant polypeptide" may include (but is not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acid deletions, insertions and / or substitutions, and additions or deletions of one or several (such as within 50, preferably within 20 or 10, and more preferably within 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, when amino acids with similar or similar properties are substituted, the function of the protein is usually not changed. For another example, adding one or several amino acids to the C-terminus and / or N-terminus usually does not change the function of the protein. The present invention also provides analogs of the polypeptide. The difference between these analogs and natural polypeptides may be differences in amino acid sequence, or differences in modified forms that do not affect the sequence, or both. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, or by site-directed mutagenesis or other known molecular biological techniques. Analogs also include analogs with residues different from natural L-amino acids (such as D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (such as β, γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

[0056] The amino terminus or carboxyl terminus of the epoxide amide synthetase of the present invention may also contain one or more polypeptide fragments as protein tags. For example, the tag may be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, ε, B, gE, or Ty1, etc.

[0057] When the purpose is to produce the epoxide amide synthetase of the present invention, in order to make the translated protein secretory (such as secreted outside the cell), a signal peptide sequence can be added to the amino terminus of the polypeptide of the present invention. The signal peptide can be cut off during the process of secretion of the polypeptide from the cell.

[0058] The active polypeptide of the present invention can be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide. The polypeptide of the present invention can be a naturally purified product, or a chemically synthesized product, or produced from a prokaryotic or eukaryotic host (e.g., bacteria, yeast, higher plants) using recombinant technology. Depending on the host used in the recombinant production scheme, the polypeptide of the present invention can be glycosylated or non-glycosylated. The polypeptide of the present invention may also include or not include an initial methionine residue.

[0059] The polynucleotide encoding the epoxide amide synthase of the present invention may be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may include a polynucleotide further including additional coding and / or non-coding sequences.

[0060] The present invention also relates to a vector comprising the polynucleotide of the present invention, a host cell produced by genetic engineering using the vector or polypeptide encoding sequence of the present invention, and a method for producing the polypeptide of the present invention by recombinant technology.

[0061] Recombinant polypeptides can be expressed or produced by conventional recombinant DNA techniques. Generally, the following steps are involved: (1) Transforming or transducing a suitable host cell with a polynucleotide encoding the polypeptide (including its conservative variant polypeptides) or a recombinant expression vector containing the polynucleotide; (2) Cultivating the host cell in a suitable culture medium; (3) Isolating and purifying the protein from the culture medium or cells.

[0062] In the present invention, the polynucleotide sequence encoding the polypeptide can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well known in the art. As long as they can replicate and be stable in the host, a variety of plasmids and vectors can be used. An important feature of an expression vector is that it usually contains a replication origin, a promoter, a marker gene and a translation control element. Preferably, the expression vector can be a eukaryotic expression vector (e.g., a yeast expression vector).

[0063] Methods well known to those skilled in the art can be used to construct expression vectors containing polynucleotides of the epoxide amide synthase of the present invention and suitable transcription / translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, etc. The DNA sequence can be effectively linked to an appropriate promoter in the expression vector to direct mRNA synthesis. In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selecting transformed host cells.

[0064] The vector containing the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence can be used to transform appropriate host cells to enable them to express proteins. The host cell can be a higher eukaryotic cell, such as a plant cell or a mammalian cell; a lower eukaryotic cell, such as a yeast cell; or a prokaryotic cell, such as a bacterial cell. Representative examples include: plant cells, such as Solanaceae plant cells, specifically tobacco cells; fungal cells, such as yeast cells; Escherichia coli cells, Streptomyces cells, Bacillus subtilis cells; cyanobacterial cells; and actinomycete cells.

[0065] The present invention also provides a recombinant cell for biosynthesis of epoxide amide, comprising: the novel epoxide amide synthase expression cassette of the present invention. The cell is used for catalyzing iso-catharanthus glycoside lactam, thereby directly synthesizing the epoxide amide.

[0066] The cell host is a production tool. Based on the teachings of the present invention, those skilled in the art can transform other host cells other than tobacco through some technical means to achieve the biosynthesis of the present invention. The host cells and production methods thus constructed should also be included in the present invention.

[0067] Application and production process

[0068] The epoxide amide synthetase or its conservative variant polypeptide of the present invention can be used to specifically and efficiently catalyze iso-catharanthus glycoside lactam, thereby directly synthesizing the epoxide amide:

[0069]

[0070] As used in the present invention, the iso-catharanthus glycoside lactam includes its derivatives, structural analogs, and isomers. The present invention also covers compounds having the same parent core structure as iso-catharanthus glycoside lactam and being able to achieve the catalytic reaction, and participating in the reaction of the present invention. The reaction can be a reaction in vitro or in an intracellular state.

[0071] In a specific embodiment of the present invention, the epoxide amide synthase from Snakeroot davidianus was expressed in tobacco, and then the substrate isocatharanthus lactam was injected, and epoxide amide was successfully synthesized in tobacco.

[0072] The application of the technical scheme of the present invention is not limited to those listed in the specific embodiments. In terms of culture conditions, the output of epoxide amide in tobacco can be improved by changing the composition, pH, culture temperature, culture time and other factors of tobacco injection buffer. In terms of biological elements, the catalytic efficiency can be improved by replacing the homologous genes or modified genes of each biological element in the synthetic pathway. On the other side of the reaction host, other host enzymes can be selected to catalyze the synthesis of epoxide amide, such as cyanobacteria, actinomycetes, Escherichia coli, yeast, etc.

[0073] Epoxide amide is a precursor substance for the biosynthesis of camptothecin. The technical solution of the present invention is suitable for scale-up, and provides a way for the large-scale synthesis of epoxide amide and further for the large-scale biosynthesis of camptothecin.

[0074] Compared with traditional plant extraction methods, the biosynthesis method of the present invention has the advantages of fast speed, less impact by external factors, etc. In the present invention, the method of cell synthesis is used to synthesize epoxide amide, which can effectively reduce the cost of separating and purifying such compounds.

[0075] The present invention also provides a kit for biosynthesis of epoxide amides, comprising: the novel epoxide amide synthetase of the present invention or its expression cassette. Preferably, the kit also includes cells for introducing the expression cassette.

[0076] As another kit for biosynthesis of epoxide amide, the kit comprises: a recombinant cell or a cell expression system, wherein the expression cassette is introduced into the recombinant cell.

[0077] As an embodiment, the kit also includes instructions for use describing the method for biosynthesis.

[0078] The positive and progressive effects of the present invention are:

[0079] It is generally believed in the art that iso-catharanthus glycoside lactam is converted into epoxide amide under the catalysis of oxidase, but the enzyme that catalyzes the synthesis of epoxide amide from iso-catharanthus glycoside lactam has not been discovered and isolated, and there is no report on the synthesis of epoxide amide using chemical methods. The present invention changes this technical prejudice and establishes a method for catalyzing the synthesis of epoxide amide in cells (such as tobacco cells) using iso-catharanthus glycoside lactam as a substrate and using the epoxide amide synthase of the present invention.

[0080] The reaction process of the present invention has less environmental pollution, and the product is easy to purify. At the same time, based on the fact that the present invention discloses that epoxide amide synthase has the function of catalyzing the synthesis of epoxide amide from iso-catharanthus glycoside lactam, epoxide amide can also be obtained by enzyme catalysis in different environments or other hosts (such as Escherichia coli, yeast, cyanobacteria, and actinomycetes).

[0081] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Experiment Guide, 3rd Edition, Science Press, or according to the conditions recommended by the manufacturer.

[0082] Example 1. Acquisition of epoxide amide synthetase gene

[0083] 16 mg of iso-catharanthus glycoside lactam and 4.9 mg of 2-(3-butynylaziridin-3-yl)acetic acid were dissolved in 0.8 mL of dimethylformamide, and then 13.2 mg of N,N'-dicyclohexylcarbodiimide and 4.0 mg of 4-dimethylaminopyridine were added in sequence. The mixture was reacted in the dark for 24 h. After cooling to room temperature, the mixture was purified by preparative high performance liquid chromatography and dried by rotary evaporation to obtain the probe ST-probe. 5 g of short snakeroot was frozen with liquid nitrogen, dissolved with 100 mL of phosphate buffer A (PBS pH 7.0: 137 mM NaCl, 10 mM Na2HPO4, 2.7 mM KCl, 2 mM KH2PO4, 5 mM DTT, and placed on ice for 0.5 h. The extract was centrifuged at 8000 rpm for 10 min to obtain the supernatant, which was then centrifuged at 12000 rpm for 10 min. The supernatant was enriched with proteins through an Amicon Ultra-15 centrifugal filter and filtered with 50 mL of phosphate buffer B (PBS The protein was washed with 137mM NaCl, 10mM Na2HPO4, 2.7mM KCl, 2mM KH2PO4) at pH 7.0, and the final protein concentration was diluted to 1mg / mL. 200μL protein and ST-probe with a final concentration of 100μM were incubated at room temperature for 1h, and UV irradiated on ice for 15min. 700μL methanol, 400μL ddH2O, and 150μL chloroform were added, mixed evenly, and centrifuged (14000rpm, 5min) to obtain the protein. 700μL methanol was added for washing and ultrasonic vibration to form a milky state. The supernatant was removed by centrifugation (14000rpm, 5min), and the protein was dissolved with 250μL buffer (50mM HEPES pH 8.0, 1% SDS), and 2.5μL azidobiotin (10mM DMSO stock solution), 2.5 μL copper sulfate (100 mM aqueous solution), 2.5 μL tris (3-hydroxypropyltriazolemethyl) amine (10 mM aqueous solution) and 2.5 μL sodium ascorbate (100 mM aqueous solution), incubate at room temperature for 1 hour, then add 700 μL methanol, 400 μL ddH2O and 150 μL chloroform in sequence, centrifuge (14000 rpm, 5 min) to obtain protein, then add 500 μL methanol to wash the protein cake, centrifuge (14000 rpm, 5 min) to remove the supernatant, add 200 μL buffer (50 mM HEPES pH 8.0, 1% SDS), filter with a 0.22 μm filter membrane after dissolution, add 30 μL streptavidin-dextran resin to each sample, rotate and incubate at room temperature for 1 hour, wash the resin 3 times with 500 μL PBS (50 mM) buffer, and then wash with 500 μL containing 0.The resin was washed three times with 5M NaCl in PBS, then washed three times with 500 μL of 50 mM trimethylamine bicarbonate solution (containing 4 M urea), centrifuged at 1000 g for 5 min, the supernatant was removed, 200 μL of trimethylamine bicarbonate solution (50 mM) containing 10 mM dithiothreitol was added, the supernatant was removed after incubation at 56°C for 30 min, 200 μL of trimethylamine bicarbonate solution (50 mM) containing 55 mM iodoacetamide (IAA) was added, and the mixture was quickly incubated at 37°C in the dark for 30 min, then washed three times with trimethylamine bicarbonate solution (50 mM) (washed on a rotator at room temperature for 15 min), 50 μL of trimethylamine bicarbonate solution (100 mM) containing 0.5 μg of trypsin was added, and enzymatic digestion was carried out at 37°C for 16 h. After centrifugation at 1000 g for 10 min, 40 μL of peptide fragments were carefully pipetted into a new 0.6 mL ep tube, dried at 80℃, 200μL ddH2O (containing 0.1% FA) was added to the sample, fully mixed and desalted, and the desalted solution was subjected to protein spectrum detection. After analysis, the candidate gene OP450-4 was obtained, and then the candidate genes OP450-1, OP450-2, OP450-3, and OP450-5 were obtained by sequence alignment of OP450-4 in the transcriptome of short snakeroot as follows:

[0084] OP450-1: nucleotide sequence SEQ ID NO: 1, amino acid sequence SEQ ID NO: 2;

[0085] OP450-2: nucleotide sequence SEQ ID NO: 3, amino acid sequence SEQ ID NO: 4;

[0086] OP450-3: nucleotide sequence SEQ ID NO: 5, amino acid sequence SEQ ID NO: 6;

[0087] OP450-4: nucleotide sequence SEQ ID NO: 7, amino acid sequence SEQ ID NO: 8;

[0088] OP450-5: nucleotide sequence SEQ ID NO: 9, amino acid sequence SEQ ID NO: 10.

[0089] SEQ ID NO: 1 (Serpentine epoxide amide synthase nucleic acid OP450-1)

[0090]

[0091] SEQ ID NO: 2 (Serpentine epoxide amide synthase protein OP450-1)

[0092] MEFFYVSLLCLFVLLVTLSLHFLFYKNKSGLSGPLPPGKTGWPMVGESLEFLSCGWKGHPEKFVFDRMAKYSSLVFKTHLLGEKAAVFCGSPGNKFLFSNENKLVQAWWPSSVDKVFPSS NQTSSKEEAIKMRKMLPNFLKPEALQRYIGIMDYIAGRHFSSGWENKDQVEVFPLCKNYTFWIASRLFVSVEDPTEVAKLLEPFNVLASGLISVPIDLPGTPFNRAIKASNEIRKQLLAI IKQRKIDLAEGKASPTQDIMSHMLTTSDENGKFMNELDVADKILGLLVGGHDTASSACTFIVKFLAELPEIYEGVYKEQMEIAKSKAPGELLNWDDIQKMKYSWNVACEVLRLAPPLQGA FREALTDFMYNGFSIPKGWKIYWSANTTHRNPDCFPEPEKFDPSRFEGSGPAPYTFVPFGGGPRMCPGKEYARLEILVFIHHVVKRFRWEKIIPDEKIVVDPMPIPAKGLPVRLYPHNDA

[0093] SEQ ID NO: 3 (Serpentine epoxide amide synthase nucleic acid OP450-2)

[0094]

[0095] SEQ ID NO: 4 (Serpentine epoxide amide synthase protein OP450-2)

[0096] MDFFYVSLLSIGVVFVSIAIHFLLYKNRSGSSANLPPGRTGLPVIGESYEFLSTGWKGYPEKFIFDRMAKYSSKVFKTHLLGEKAFVFCGAAGNKFLFSNENKLVQSWWPSSVDKVFPS SNQSSSKEEAIKMRKMLPNFLKPEALQRYIGIMDQIAQRHFTSGWENKDQVEVFPLCKNYTFWLASRLFVSVEDPTEVAKLLEPFNVLASGLISVPIDLPGTPFNKAIKASNQVRKQLVA IIKQRKIDLTEGKATPTQDIMSHMLTTSDENGKFMNELDVADKILGLLIGGHDTASSACTFIVKFLAELPEIYEGVYKEQMEIAQEKAPGEFLNWEDIQKMKYSWNVACEVLRLAPPLQG AFREALSDFMFNGFTIPKGWKIYWSANSTHRNQECFPEPLKFDPSRFDGSGPAPYTFVPFGGGPRMCPGKEYARLEILVFMHNLVKRFRWEKIIADEKIVVDPMPIPAKGLPVRLYPHKA

[0097] SEQ ID NO: 5 (Serpentine epoxide amide synthase nucleic acid OP450-3)

[0098]

[0099] SEQ ID NO: 6 (Serpentine epoxide amide synthase protein OP450-3)

[0100] MEVLLLCMLSLVVIVLSLFILSYFSKNNGIKLPPGSTGWPILGENLKLASGPEKFIQKRMELYSEKVFKTSIVGEKMAVFCGAAGNKFFFTNENNLLTTWWPESFIKPMSFPSSKEKS MKEITGIHRSFTEEFLRPESLKQYIPVMDSMAREQLETDWAPFDEVKVHPLSKKYTFSLACKLFLGVEDPKLVKLLSESFNIATTGMFSVPIDLPGTAYNRAMKGGKVVKDQFLRIIK ERKKELLERNAAEGFDFLTRMLLRTDESARFMTETEIGNNIIGLLIAGYDTTSAGVSFVLNYLAELPDIYNKVYKEMMEIAKSKGPRELLNWNDISKMKYSWNVACEALRLTPPAQGA FREAITDFTYAGFTIPKGWKTFWSVYSTHKNPKYFPDPEKFDPSRFEGTGPAPYTYVPFGGGSRMCPGREYARLEILVFMYNVLRRFRLEKLIQDERVIYHLSAIPEDGLPVRLIPQRK

[0101] SEQ ID NO: 7 (Serpentine epoxide amide synthase nucleic acid OP450-4)

[0102]

[0103] SEQ ID NO: 8 (Serpentine epoxide amide synthase protein OP450-4)

[0104] MEALVQYLLLPLVLFPLSIAVIVFFKKRGSSSLKVPPGTTGWPLLGENIHFALLGSQKFVSDRMKKYSPEFFQTSLMGEKMAIFCGPQGNKFLFTNENKLLTSWWPQSMKKALLFPEYVE DSLKEVSALKRSFMHDILKPEALKQYIPVMDALAREHIEQEWTPNKEVKVFPLSKKYTFDLACRLFMSIEDPAHIKKLADPFHLVTNGMFSVPIDLPGTAYNGAIKGGKMVRAELLKII RDRRKELTESKETEGRDLLSRMLLVTDEEGKFLSEMEISNNIIGLLVASYETTSSAVTFVLKHLAELPHIYDEVFKEQMAIAKTKGPDELLTWEDIEKMKYSWNVARESLRVTPPAQGA FRESVTDFIYAGFTVPKGWKTFWTVHTTHKNPKYFPEPEKFDPSRFEGSGPMPYTFVPFGGGPRMCPGKEYARLEVLVFMYNVVRNFKLEKSIANEKIAYHASPTPMKGLPVRLYPHGN

[0105] SEQ ID NO: 9 (Serpentine epoxide amide synthase nucleic acid OP450-5)

[0106]

[0107] SEQ ID NO: 10 (Serpentine epoxide amide synthase protein OP450-5)

[0108] MLPNFLKPEALQRYIGIMDSIAGRHFSSSWENKGQVEVFPLCKNYTFWIASRLFVSVEDPTEVAKLLEPFGLLASGIISVPVDLPGTPFNKAIKASNEIRKRLLVIIKQRKIDLAEGKASPTQDIMSHMLTTSDENGKFMHELDVADKILGLLIGGHDTASSACTFIVKFLA ELPEIYDGVYKEQMEIAKSKAPGELLNWNDIQNMKYSWNVACEVMRLAPPLQGAFREALTDFMYNGFSIPKGWKLYWSANTTHKNPECFPEPEKFDPSRFEGSGPAPYTFVPFGGGPRMCPGKEYARLEILVFMHHLVKRFRWEKIIPDEKIVVDPLPSPAKGLPIRLYPRNA

[0109] Using the cDNA of D. brevis as a template, PCR amplification was performed using forward primers and reverse primers targeting the corresponding nucleic acid sequences of each enzyme. The corresponding primers are shown in Table 1.

[0110] Table 1

[0111]

[0112] A small fragment containing the epoxide amide synthase gene reading frame and homology arms was obtained by PCR amplification and agarose gel recovery.

[0113] Example 2. Expression of epoxide amide synthetase

[0114] The pCambia2301 vector was cut with the fast-cutting enzymes BamHI and SalI, and then a large fragment of the pCambia2301 vector containing the BamHI and SalI recombination sites was obtained by agarose gel recovery; the large fragments of the epoxide amide synthase gene from short snakeroot were recombined into the pCambia2301 vector by homologous recombinases to obtain a recombinant expression vector (recombinant product) with the target gene inserted.

[0115] The recombinant product was transferred into Escherichia coli TOP10, and the plasmid was extracted from the positive Escherichia coli TOP10 to obtain a plasmid carrying the target gene; then the plasmid carrying the target gene was transferred into Agrobacterium (GV3101) to obtain Agrobacterium containing the epoxide amide synthase gene derived from short snakeroot.

[0116] Fermentation was performed with 10 mL of liquid LB medium at a rotation speed of 250 rpm and a temperature of 28° C. When the OD600 of the fermentation liquid was 1, the fermentation liquid was centrifuged at 4000 rpm for 10 min.

[0117] The fermentation product was obtained by removing the supernatant to obtain the bacterial cells, which were evenly resuspended in 10 mL of MES buffer (pH 5.6, 10 mM 2-(N-morpholine)ethanesulfonic acid, 10 mM MgCl2, 0.1 mM acetosyringone) and placed at room temperature for 2 h.

[0118] The bacterial solution was drawn up with a syringe without a needle and injected into the back of one-month-old tobacco, which was then cultured (16 h of light and 8 h of darkness per day) for 4 days to obtain tobacco expressing epoxide amide synthase. The Agrobacterium-treated group with only the pCambia2301 plasmid was used as the blank control.

[0119] Example 3: Synthesis of epoxide amides from isovinca glycoside lactam catalyzed by epoxide amide synthetase

[0120]

[0121] Isovinca glycoside lactam was dissolved in MES buffer to obtain a final concentration of 0.1 mM, and then injected on the back of tobacco expressing epoxide amide synthase for catalytic synthesis. Tobacco expressing empty plasmid pCambia2301 served as blank control. After 3 days, the tobacco leaves were ground in a mortar, 2 mL of methanol was added to each leaf, and the grinding liquid was centrifuged (12000 rpm) for 10 min to obtain the supernatant. 100 uL of the supernatant was taken for mass spectrometry analysis.

[0122] Epoxide amide synthase catalyzes the Figure 2 The results showed that the products of the five plant epoxide amide synthase reaction groups were the same, with a peak time of 3.77 min, which was consistent with the peak time of the purified epoxide amide standard. The product molecular weight was 515.2006, which was not significantly different from the purified epoxide amide standard 515.2001. No corresponding new peak appeared in the blank control group.

[0123] The mass spectrometry results of the products generated by the epoxide amide synthase of Snakeroot sphaerocarpon are as follows Figure 3The results showed that the ion fragments of the product were 353, 335, 283, and 265, which were consistent with the purified epoxide amide standard, indicating that epoxide amide was generated in tobacco under the catalysis of epoxide amide synthase from D. pyrifera. In the future, epoxide amide synthase from D. pyrifera can be used to catalyze the synthesis of epoxide amide from isocatharanthus glycoside lactam.

[0124] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims. At the same time, all the documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference separately.

Claims

1. A method for synthesizing epoxide amides, comprising: The epoxide amide is synthesized by catalyzing vinca glycoside lactam with epoxide amide synthetase; wherein the epoxide amide synthetase is a polypeptide having an amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10 or a conservative sequence variant thereof.

2. The method according to claim 1, characterized in that The method is carried out intracellularly; preferably, an expression cassette of epoxide amide synthetase is introduced into the cell to express epoxide amide synthetase, thereby catalyzing the isovinca glycoside lactam and synthesizing the epoxide amide.

3. Use of epoxide amide synthetase for catalyzing iso-catharanthus glycoside lactam and synthesizing the epoxide amide, wherein the epoxide amide synthetase is a polypeptide having an amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10 or a conservative sequence variant thereof.

4. The use according to claim 3, characterized in that The catalysis is carried out in cells; preferably, an epoxide amide synthetase expression cassette is introduced into the cells to catalyze iso-vinca glycoside lactam and synthesize the epoxide amide; preferably, the expression cassette is contained in a recombinant expression vector.

5. An isolated epoxide amide synthetase, which is a polypeptide having an amino acid sequence as shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10, or a conservative sequence variant thereof.

6. The epoxide amide synthetase according to claim 5, characterized in that The conservative variant polypeptides include: (1) A polypeptide having the function of synthesizing epoxide amide formed by substitution, deletion or addition of one or more amino acid residues of the polypeptide of the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:10; (2) a polypeptide having an amino acid sequence that is more than 80% identical to the polypeptide shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:10, and having the function of synthesizing epoxide amide; or (3) A polypeptide formed by adding a tag sequence to the N-terminus or C-terminus of a polypeptide having an amino acid sequence as shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10, or by adding a signal peptide sequence to its N-terminus and retaining its function.

7. An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: (1) A polynucleotide encoding the epoxide amide synthetase according to claim 5 or 6; (2) a polynucleotide complementary to the polynucleotide (1); Preferably, the polypeptide corresponding to the amino acid sequence shown as SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:10, the nucleotide sequence of the polynucleotide is shown as SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:

9.

8. An expression construct comprising the polynucleotide of claim 7.

9. A recombinant cell comprising an exogenously introduced epoxide amide synthetase expression cassette; the epoxide amide synthetase is a polypeptide having an amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10 or a conservative sequence variant thereof.

10. A method for preparing the recombinant cell according to claim 9, comprising: The epoxide amide synthase expression cassette is introduced into the cell to obtain the recombinant cell; the epoxide amide synthase is a polypeptide of the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10 or a conservative sequence variant thereof; preferably, the expression cassette is contained in a recombinant expression vector.

11. A kit for biosynthesis of epoxide amides, comprising: The epoxide amide synthetase or its expression cassette according to claim 5 or 6; or The recombinant cell of claim 9.

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