CN113930400A - Securinega suffruticosa derived oxidase and application thereof - Google Patents

Abstract

The invention discloses an oxidase SEQ ID NO. 2 derived from securinega suffruticosa, which can catalyze L-ascorbic acid/dehydroascorbic acid to carry out an oxidative condensation reaction with securinega suffruticosa alkali to prepare securinega suffruticosa alkaloid flueaffine A and analogues thereof and has research and development values.

Description

Securinega suffruticosa derived oxidase and application thereof

Technical Field

The invention belongs to the field of bioengineering, and particularly relates to a securinega suffruticosa derived oxidase FsBBE and application thereof in preparation of securinega suffruticosa alkaloid fluesuffine A.

Background

Securinega suffruticosa, known as Fluegea suffrutinosa (Pall.) Baill, is a plant of phyllanthus of Euphorbiaceae, is widely distributed in China, and is not found in China except northwest. Securinega suffruticosa can be used for treating nervous related diseases such as paralysis, neurasthenia, lethargy, etc. The securinega suffruticosa contains abundant securinega alkaloid, is considered as a biological active component of a securinega plant, and comprises securinine (securinine) and securinine (allosecurinine), wherein the securinine is a medicament used for treating sequela of poliomyelitis, active compounds with novel structures are separated from the securinega suffruticosa in recent years, and the new compounds have multiple activities of resisting tumors, viruses, bacteria, central nerves and the like. The research on the alkaloid in the securinega suffruticosa can discover more bioactive components, and the analysis on the synthetic pathway of the alkaloid in the securinega suffruticosa can find some enzymes with new functions and new biocatalysis tools, so that a foundation is laid for synthesizing the securinega suffruticosa alkaloid by a synthetic biology method, and the plant resource of the traditional Chinese medicine can be more fully developed and utilized.

Disclosure of Invention

Some alkaloid components in the securinega suffruticosa are extracted, separated and identified, and the securinega suffruticosa alkaloid with a novel structure is obtained by separation, has a molecular structure shown in the following formula 1 and is named as fluebuffine A:

the ABCD ring part in the molecular structure of the compound 1 is very similar to the structure of allosecurinine/securinine, and is shown in the following formula.

It is speculated that the ABCD loop moiety is likely to be derived from allocoresine or securinine, the 6-carbon moiety in the structure in which the FG loop is highly oxidized, is likely to be derived from vitamin C or L-ascorbic acid (L-AA), and thus compound 1 is likely to be a vitamin C-modified natural product. The biosynthetic pathway of compound 1 is inferred to be the following pathway.

Tyrosine (L-tyrosine) and lysine (L-lysine) are polymerized into alloneuronine/securinine after multi-step enzyme catalytic modification, and then the alloneuronine/securinine and L-ascorbic acid (L-AA) are polymerized into the compound 1 under the catalytic action of one or more oxidase (oxidase) in one step or multiple steps.

To verify the above speculation, we isolated an oxidase from the plant of securinega suffruticosa and its coding gene, and found that it should belong to the BBE family of oxidoreductases through sequence analysis and comparison, so named as FsBBE, which can catalyze the oxidative condensation reaction of L-ascorbic acid or dehydroascorbic acid (DHA) and securine to obtain compound 1.

Accordingly, in a first aspect the present invention provides an isolated polypeptide selected from the group consisting of:

(a) a polypeptide having the amino acid sequence of SEQ ID NO: 2:

MNPLKHSSSTPLVFVLLTVCSCATSVTIPELFFQCLSNTTTTSTSIFNVLYTPRNTSYTSILESRIQNLRFNTTDTPKPLAIVTPLDASHIQATIICARKHNLQIRIRSGGHDYEGLSYVSPLPFVVLDLINLRNITVDVENRVAWVGCGATLGEFYYRIAEKTRTLAFPAGACPTVGVGGHFSGGGYGYLLRKFGLAADNILDASLVDVNGRILDRASMGEDLFWAIRGGGGNSFGVVIAWKVNLVPVPSTLTSFKVSKSLEQNTMIQLLNKWQYVANKLPDELSMFAVVSKKNSTISVKFYSLYVGGIDSLLPLMEERFPELGLKRADCNEMSWIESAVSFAGYASNTSLDVLLNHTNNYEIASGRFKGKSDFVKEPVPEAALEGLLKWLSDKDITNAAIYMVPLGGKMGEITETSIPFPHRAGNLYLLAYYVKWEGQGTEAAQKPLSWIRKGYKYMAPYVSKNPREAHLNDRDLDIGTNNISGNTSYEQASIWGTKYFKNNFDRLVRVKTSVDPSDFFRNEQSVPPLLS(SEQ ID NO:2)；

(b) 2 by substitution, deletion or addition of one or more amino acid residues, and has (a) polypeptide function;

(c) a polypeptide derived from (a) having a homology of 95% or more, preferably 98% or more, more preferably 99% or more with the polypeptide sequence defined in (a) and having the function of the polypeptide of (a); or

(d) A derivative polypeptide of the polypeptide sequence in (a) or (b) or (c) is contained in the sequence.

In a second aspect the present invention provides an isolated polynucleotide selected from the group consisting of:

(A) a polynucleotide encoding the polypeptide of claim 1;

(B) a polynucleotide encoding a polypeptide having the amino acid sequence shown in SEQ ID NO. 2;

(C) the polynucleotide with the nucleotide sequence shown as SEQ ID NO. 1;

(D) polynucleotide whose nucleotide sequence has homology of 95% or more, preferably 98% or more, more preferably 99% or more with the nucleotide sequence shown in SEQ ID NO. 1;

(E) a nucleotide sequence complementary to the nucleotide sequence of any one of (A) to (D).

Preferably, the polynucleotide is SEQ ID NO 1.

Another aspect of the present invention provides a vector comprising the above polynucleotide, and a microorganism transformed with the vector.

The microorganism can be selected from Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, yarrowia lipolytica, and Bacillus subtilis. Preferably E.coli BL21(DE 3).

The fourth aspect of the invention provides the use of the polypeptide or the microorganism in the preparation of securinega suffruticosa alkaloid (flueuffine A) and compound 2 (flueuffine B) shown in formula 1.

In the preparation of compound 1, compound 1 is prepared by an oxidative condensation reaction catalyzed by the above-mentioned polypeptide using, for example, L-ascorbic acid (L-AA) or dehydroascorbic acid (DHA) and securinine (allosecurinine) as substrate raw materials.

Specifically, the substrate material is selected from L-ascorbic acid and securinega suffruticosa alkaloid or dehydroascorbic acid and securinega suffruticosa alkaloid.

In the preparation of compound 2, compound 2 is prepared by, for example, oxidation-condensation reaction using isoascorbic acid (isoascorbyl acid) and securinine (allosecurinine) as substrate raw materials under the catalysis of the above-mentioned polypeptide.

Specifically, the substrate material is selected from isoascorbic acid and securinega suffruticosa alkali.

The polypeptide SEQ ID NO. 2 disclosed by the invention can catalyze the oxidation condensation of securinega suffruticosa alkaloid (allosecurinine) and L-ascorbic acid (L-AA) or dehydroascorbic acid (DHA) to prepare a novel securinega suffruticosa alkaloid fluensuine A; and can catalyze the securinega suffruticosa alkali and the isoascorbic acid to carry out oxidative condensation to prepare a novel compound flueuffine B, lays a foundation for analyzing the synthetic biology of the alkaloid in the securinega suffruticosa, promotes the development and utilization of new natural medicines, and has further research and development prospects.

Drawings

Fig. 1 shows a photograph of a culture of a sterilized seedling of securinega suffruticosa and a photograph of feeding L-AA precursor to securinega suffruticosa leaves.

FIG. 2 shows the results of the contents of different L-AA precursor compounds 1 fed to Securinega suffruticosa and isotopically labeled L-ascorbic acid (1-¹³C) Statistical plot of isotopic abundance of compound 1. FIG. A: results of feeding the contents of the different substrate compounds 1, the ordinate is relative content; and B: i: feeding non-isotopically labeled L-ascorbic acid (1-¹³C) Isotopic abundance of compound 1; ii: feeding isotopically labelled L-ascorbic acid (1-¹³C) Isotopic abundance of compound 1; iii: 3 and L-AA in D₂Isotopic abundance of compound 1 obtained by the reaction in O.

FIG. 3 is a statistical graph showing the relative content of Compound 1 in Securinega suffruticosa leaves induced by MeJA for various periods of time. Wherein the ordinate is relative fresh weight content, water is water, leaf is leaf, root is root, stem is stem.

FIG. 4 is a graph showing the results of Q-TOF detection of proteins in Securinega suffruticosa leaves. Wherein the abscissa is the retention time. The left side shows the results of extracting EIC (390.1183), i: the results of the activities of the total protein of the leaves of the securinega suffruticosa, allosecurinine and L-AA; ii: the results of the activities of the total protein of the securinega suffruticosa leaf, allosecurinine and DHA; iii: the activity results of the purified FsBBE protein, allosecurinine and L-AA; iv: the activity results of the purified FsBBE protein, allocorenin and DHA; v: the activity results of the purified FsBBE protein, allosecurinine and L-AA; vi: the activity results of the purified FsBBE protein, allocoresine and isoascorbic acid; vii: compound 1 standard. The right side is the research result of the FsBBE mechanism, i and ii are graphs of EIC 216.1019 extracted by the reaction of purified FsBBE protein and allocoresine and the reaction of total protein of tobacco leaves injected with FsBBE gene and allocoresine respectively; iii, iv and v are graphs of the reaction extraction 390.1183 results of enamine intermediate 3 and L-AA, DHA and isoascorbic acid, respectively; vi and vii are standards for compounds 2 and 1, respectively.

FIG. 5 shows the photograph of SDS-PAGE gel electrophoresis silver staining analysis of the active components after the total protein of Securinega suffruticosa leaves passes through the ion exchange column.

FIG. 6 shows a photograph of an SDS-PAGE gel electrophoresis silver stain analysis of the active fraction after molecular sieving. The bands marked FS-F0-11-33-1 and FS-F0-11-34-1 are bands of the target protein.

FIG. 7 shows a photograph of SDS-PAGE gel electrophoresis silver staining of candidate active proteins purified from tobacco, with FsBBE protein bands marked by arrows.

Figure 8 shows the absolute configuration of compounds 1 and 2.

FIG. 9 shows the proposed synthetic mechanism for compound 1 and compound 2.

Detailed Description

In order to analyze and identify alkaloid components in securinega suffruticosa, branches and leaves of securinega suffruticosa are extracted by organic solvents such as ethanol and ethyl acetate, and the obtained total alkaloids are subjected to silica gel column chromatography and semi-preparative high performance liquid chromatography for separation to obtain compound 1. Using spectral analysis including high resolution electrospray ionization mass spectrometry (HRESIMS),¹H and¹³C-NMR, in combination with two-dimensional nuclear magnetic resonance, Infrared (IR), etc., determines its molecular structure, and determines its absolute configuration by X-ray diffraction.

The compound 1 comprises the skeleton structure of allosecurine (securinine) or securinine (securinine), and should be the oxidation condensation product of allosecurinine/securinine and L-ascorbic acid/dehydroascorbic acid, and the oxidation condensation process is carried out under the catalysis of oxidase FsBBE.

In order to allow the oxidase FsBBE to be applied to similar catalytic reactions, expression of the protein by a microorganism is the best method for producing the enzyme.

Since the amino acid sequence of the oxidase FsBBE of the present invention is clear, the genes encoding the same, expression cassettes and plasmids containing the genes, and transformants containing the plasmids are easily obtained by those skilled in the art. These genes, expression cassettes, plasmids, and transformants can be obtained by genetic engineering construction means well known to those skilled in the art.

The above-described transformant host may be any microorganism suitable for expressing the enzyme SEQ ID NO. 1, including bacteria and fungi. Preferably the microorganism is selected from the group consisting of Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, yarrowia lipolytica, Bacillus subtilis. More preferably E.coli BL21(DE 3).

When used as a biocatalyst for the production of alkaloids of similar structure such as flueuffine A, the oxidase FsBBE of the present invention may be in the form of an enzyme or in the form of a bacterial cell. The enzyme forms comprise free enzyme and immobilized enzyme, including purified enzyme, crude enzyme, fermentation liquor, enzyme immobilized by a carrier and the like; the form of the thallus comprises a viable thallus and a dead thallus.

The isolation and purification of the oxidase FsBBE of the present invention, including immobilized enzyme preparation techniques, are also well known to those skilled in the art.

The present invention will be described in further detail with reference to specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.

The addition amount, content and concentration of various substances are referred to herein, wherein the percentage refers to the mass percentage unless otherwise specified.

In the examples herein, if no specific description is made about the reaction temperature or the operation temperature, the temperature is usually referred to as room temperature (15 to 30 ℃).

EXAMPLE 1 isolation and characterization of Compound 1

1.1 extraction and isolation of Securinega suffruticosa alkaloid

Stems and leaves of securinega suffruticosa, purchased in south-south of Henan Yang in 2017 in 9 months, identified by Yangye hong researcher at Shanghai mountain plant science research center of Chinese academy of sciences.

Grinding the branches and leaves of Securinega suffruticosa (L.) Securinega Suffruticosa (L.) Securinega) S.C. and dry weight of leaves of 1.8 kg), repeatedly extracting with 5L ethanol for 4 times, distilling and concentrating ethanol phase to obtain ethanol extract, re-suspending the ethanol extract with 1L diluted hydrochloric acid aqueous solution with pH of 6.0, extracting with ethyl acetate of equal volume for three times, adjusting pH of water layer with ammonia water to 8.0, and collecting the water layerExtracting with equal volume of ethyl acetate for three times, mixing ethyl acetate phases, distilling and concentrating to obtain total alkaloid 27.0g in terms of CH₂Cl₂The mobile phase of MeOH is 100:0,30:1,20:1,15:1,10:1,7:1,4:1,2:1,1:1 and 0:1, the silica gel column is used for separation, TLC detection is carried out, similar fractions are combined, and 8 components are obtained: F1-F8, fraction F5 was separated again with semi-preparative high performance liquid chromatography on a Luna C18 semi-preparative column (250X 10mm,5 μm) with mobile phase: methanol: 0.1% formic acid water is 5:95-25:75, 0-10 min; 25:75-25:75, 10-15 min; 25:75-95:5, 15-25 min; gradient eluting for 95:5,25-30min, separating to obtain compound 1 (t)_R22.5 min). Compound 1 was dissolved in deuterated chloroform for nuclear magnetic identification in the solvent ethyl acetate: the methanol was slowly evaporated at room temperature under the condition of 7:1 to obtain a single crystal of compound 1, and the absolute configuration of the single crystal was determined by X-ray diffraction.

1.2 isolation and Structure characterization of Compound 1

The compound 1 obtained by separation is light yellow solid, and HRESIMS (high resolution electrospray ionization mass spectrometry) M/z [ M + H ]]⁺390.1183, shows its molecular formula C₁₉H₁₉NO₈The unsaturation degree was 11. IR spectrum showed hydroxyl group (3452 cm)^-1) Carbonyl group (1745 cm)^-1) And double bonds (2943 cm)^-1、1441cm^-1And 1378cm^-1) And (4) absorbing.¹H and¹³the C-NMR spectrum combined with the two-dimensional nuclear magnetic spectrum showed (see Table 1) that Compound 1 had two ester or amide carbonyl groups (. delta.) (_C171.4,174.3), 1 sp²Quaternary carbon delta_C163.7, 3 sp²Methine group (. delta.)_C/H148.8/7.05,122.4/6.57,111.3/5.86), 4 lower fields of sp³Quaternary carbon (. delta.)_C112.3,101.5,93.1,80.4), 4 sp³Methine group (. delta.)_C/H90.9/5.05,74.4/4.52,56.5/3.92, 37.0/2.11)), one of which is methine in the lower field, 5 sp³Methylene (table 1.3.1). Comparing the data with reported alkaloid nuclear magnetic data separated from securinega suffruticosa, the compound 1 contains allosecurinine or mixed source dipolymer of securinine framework structure. Detailed analysis of the two-dimensional nuclear magnetic spectrum of compound 1 shows that the planar structure of compound 1 is shown in formula 1, and compound 1 has 8 handsAnd 4 of the carbon atoms are quaternary carbons. After several attempts we fortunately obtained a single crystal that was able to determine the quality of the absolute configuration of the compound, and single crystal diffraction results showed that the compound was in the 2R,3R,7S,9S,2 'S, 3' R,4 'R, and 6' S configurations. We named compound 1 fluesuffine a.

TABLE 1 preparation of Compound 1¹H spectrum and¹³c NMR data

The biosynthetic pathway of compound 1 was analyzed and validated as follows.

Example 2 culture of aseptic seedlings of Securinega suffruticosa

Seed was placed at 0.1% v/w

20, slowly oscillating for 5min at 90rpm of a shaking table, and washing with distilled water for 3 times; then placing the seeds into ethanol with the mass fraction of 70% for surface sterilization for 1min, immediately transferring to 0.5% v/v sodium hypochlorite solution and slowly oscillating at 90rpm for 12 min; then washing the seeds with sterile water in a super clean bench for 3 times, putting the seeds into the sterile water overnight, pouring the sterile water in the super clean bench, sucking the excess water on the surfaces of the seeds with sterile filter paper, putting the seeds into a sterile and moist filter paper of a sterile flat plate, standing overnight at 4 ℃, and putting the seeds into a 24 ℃ constant temperature incubator to be cultured in a dark place. After the seeds are cultured on the flat plate for about one week, the seeds begin to germinate, the seeds are continuously cultured for 3 days at 24 ℃ for 16 h/day under illumination, aseptic seedlings grow into aseptic seedlings, the roots of the aseptic seedlings are cut off, the aseptic seedlings with the roots cut off are transferred to a PGMO5 culture medium, and the aseptic seedlings are continuously cultured for about 3-4 weeks in a constant temperature incubator at 24 ℃ for 16 h/day under illumination, as shown in figure 1.

PGMO5 medium: 2.2g MS culture medium, 10g sucrose, 4g phytogel and water to make 1L, adjusting pH to 5.8 with NaOH solution, and sterilizing at 121 deg.C for 20 min.

Example 3L-AA precursor and isotopically labeled L-ascorbic acid (1-¹³C) Feeding experiment of

Feeding experiment of L-AA precursor including D-glucose (D-glucose), D-mannose (D-mannose), L-galactose (L-galactose), L-galactono-1,4-lactone (L-galactono-1,4-lactone), L-ascorbic acid (L-ascorbic acid) and the like was performed with sterilized leaf of Securinega suffruticosa grown for about 5 weeks, then three seedlings of Securinega suffruticosa were taken out from the bottle (see FIG. 1), the leaves of sterilized seedlings were cut off (the lowermost and uppermost leaves were discarded), and the leaves were cut off from the middle of the leaves, the leaves were immersed in an aqueous solution (concentration of 5mM, as shown in FIG. 1) of the precursor feeding compound, three replicates were performed for each of the precursor feeding compound, water was used as a control group, and the sterilized leaf of Securinega suffruticosa was completely immersed in the aqueous solution of the precursor compound during feeding, shaking at 90rpm, irradiating at 24 deg.C for 72 hr, and culturing in constant temperature incubator for 72 hr. Taking out the leaves after 72h, sucking excessive water by using absorbent paper, weighing, putting into a 1.5ml EP tube, freezing and grinding by using liquid nitrogen, adding 1ml of methanol (HupA with the concentration of 50nM is an internal standard) extracting solution containing 0.1% formic acid into each 100mg of wet weight sample in equal proportion, carrying out ultrasonic extraction for 10min, centrifuging for 10min at 17000g, filtering supernatant by using a 0.2 mu M filter membrane, detecting Q-TOF, calculating the relative content of the compound 1, and calculating the method: (1/hupzine a 50x10-6 x 1x10-3) 389.36/100 (compound 1 and hupzine a (huperzine a) represent peak areas from which EIC 390.1183 and 243.1497 were extracted when mass spectrometrically identified. Content results for feeding different substrate Compound 1 and feeding isotopically labeled L-ascorbic acid (1-¹³C) The isotopic abundance of compound 1 is shown in figure 2.

As can be seen from FIG. 2, the yield of compound 1 was significantly improved after the leaves of Securinega suffruticosa were fed with L-galactose (L-galactonase), L-galactono-1,4-lactone (L-galactono-1,4-lactone) and L-ascorbic acid (L-ascorbyl acid), indicating that these added compounds are all precursors for the biosynthesis of compound 1, proving our inference about the biosynthetic pathway of compound 1.

Example 4 treatment of Securinega suffruticosa seedling with methyl jasmonate and measurement of the content of Compound 1 in the seedling leaves after the treatment

About one month old seedlings of securinega suffruticosa were grown, at which time about 7 leaves were grown (see fig. 1), 8 plants were placed in each bottle, sprayed with a 50 μ M aqueous solution of Methyl Jasmonate (Methyl Jasmonate, MeJA), treated for 24, 48 and 72 hours, respectively, and water (water) was used as a blank control group (sprayed every 12 hours), for 4 experiments, each of which was repeated three times. The method for measuring the content of the novel compound in the seedling leaves is the same as that in example 3. The content of compound 1 in cacumen Securinegae Suffruticosae is shown in figure 3.

As can be seen from fig. 3, treatment of securinega suffruticosa seedlings with methyl jasmonate MeJA can increase the biosynthesis amount of compound 1, with the longer the treatment time, the higher the yield of compound 1.

Example 5 MeJA Induction experiment of sterile seedlings of Securinega suffruticosa and transcriptome sequencing of MeJA induced leaves at different times

Example 4 seedlings leaves at 0h (water blank), 24h and 72h after MeJA treatment were subjected to second generation transcriptome sequencing using Illumina HiSeq as the instrument^TMAnd filtering the obtained data to obtain clean data, and splicing the obtained clean data by using Trinity to obtain 145153 transcripts.

The obtained transcript is subjected to coding sequence CDS (coding sequence) prediction, and the CDS prediction method comprises the following steps: 1. comparing Gene according to the priority sequence of an NR protein library and a Swissprot protein library, if the comparison is up, extracting ORF coding frame information of a transcript from the comparison result, and translating a coding region sequence into an amino acid sequence (according to the sequence of 5'- > 3'); 2. for sequences without aligned NR protein library, Swissprot protein library or sequences with results not predicted on alignment, the open reading frame ORF (open reading frame) is predicted by using estscan (3.0.3) software, and the nucleic acid sequence and amino acid sequence coded by the part of genes are obtained.

Example 6 extraction and Activity experiment of Securinega suffruticosa aseptic seedling leaf Total protein

After the sterilized seedlings of Securinega suffruticosa (Securinega suffruticosa) growing for about one month were cut, the leaves were weighed, and the wet weight of the seedlings was 8.2g, and the seedlings were subjected to nitrogen freeze-grinding, 41ml of extraction buffer A (100mM NaPi, pH 7.4,5mM sodium bisulfite, 5mM dithiothreitol, 1mM EDTA, 10% (v/v) glycerol, 1% (w/v) PVP, 4% (w/v) PVPP) was added thereto, incubated on ice, gently shaken up and down several times every 15 minutes, after 1 hour, centrifuged at 5000g at 4 ℃ for 30min, the supernatant was desalted through a pD10 column and replaced with buffer B (10mM Tris-HCl, pH 7.5). After the protein was extracted, the concentration of the protein was measured by the Bradford method, and the concentration of the protein was 3.5 mg/ml.

50 mu L of desalted protein is added with allocerinine/securinine (0.5 mu L, the initial concentration is 100mM) and L-ascorbic acid (L-ascorbyl acid, L-AA)/dehydroascorbic acid (DHA) (0.5 mu L, the initial concentration is 100mM), the control group is heat-inactivated protein, the rest are the same, after the protein is placed in a shaking table at 30 ℃ for 2h, the reaction is stopped by adding equal volume of methanol, 17000g is centrifuged for 10min, and after the filter membrane of 0.2 mu M is filtered, Q-TOF (four-rod-time of flight mass spectrometry) is detected. The results are shown in FIG. 4.

As can be seen from the left graph in FIG. 4, the total protein of Securinega suffruticosa leaf can catalyze the oxidative condensation of allosecurinine and L-AA to obtain the compound 1 (see i in the graph), and can catalyze the oxidative condensation of allosecurinine and DHA to obtain the compound 1 (see ii in the graph).

Example 7 fractionation and protein Mass Spectrometry verification of Total protein of Securinega suffruticosa leaf

The extraction method of total protein of Securinega suffruticosa was the same as that in example 6, and about 175.0mg of desalted total protein was fractionated with Hitrap Q HP column (GE), and eluted by gradually increasing NaCl concentration in buffer B at a flow rate of 2.5 ml/min. The F0 protein fraction, which did not bind to the column, was shown to be active by activity testing (activity testing method was the same as total protein testing method, data not shown). Therefore, the protein fraction F0 which did not bind to the column was concentrated using a 10kDa ultrafiltration tube, and buffer C (10mM MES, pH 6.5) was gradually added to the ultrafiltration tube to replace the buffer.

The buffer-exchanged fraction F0 was then fractionated with Hitrap SP HP column (GE), eluting with buffer C at increasing NaCl concentration at a flow rate of 1.0 ml/min. One component was received every 1.5 ml. A total of 40 components F0-F1-F0-F40 were received. And activity test was performed on each fraction (activity test conditions were the same as F0, EIC (390.0, [ M + H ])]⁺) And the peak areas obtained by extraction were extracted to judge the strength of the activity of each component), F0-F10 and F0 were foundThe F11 fraction was most active (data not shown), while the active fractions F0-F9-F0-F17 were analyzed by SDS PAGE (12% gel) silver staining, and multiple protein bands were still present in fractions F0-F10 and F0-F11, requiring further fractionation (FIG. 5).

The fraction F0-11 with the best activity separated by Hitrap SP HP column was concentrated to below 500. mu.L with a 10kDa ultrafiltration tube, and further separated and purified with Superdex 200 Increate 5/150GL molecular sieve column at 0.3ml/min, and eluted with buffer B, receiving one fraction every 0.5ml, and receiving 60 fractions in total (F0-11-1-F0-11-60). The activity test showed that the components F0-11-33 and F0-11-34 were the most active (the activity test conditions were the same as those of the F0 component, and the data of the test results were not shown), and the active components (F0-11-29-F0-11-37) were analyzed by SDS PAGE (10% gel) silver staining (FIG. 6).

Corresponding to the activity test result, the red marked band in FIG. 6 is the target protein band, so that the mass spectrometric identification of the enzyme-cleaved protein in the gel is carried out on F0-11-33-1 (marked as the target band in FIG. 6) and F0-11-34-1 (marked as the target band in FIG. 6). Cutting the glue into 1mm multiplied by 1mm colloidal particles, putting the colloidal particles into a 1.5ml centrifuge tube, and simultaneously and respectively carrying out the following operations on the samples: 200 μ L ddH₂O rinse 3 times. Adding silver dye decolorant (100mM NaS)₂O₃And 30mM potassium ferricyanide) was incubated at 37 ℃ for 30mins to decolorize, and the operation was repeated several times until the color had gone. Add 200. mu.L of ddH₂And O incubation for 5min, and repeating the operation once. After 5min incubation 100. mu.L of 50% AcN was added, centrifugation was carried out, the supernatant was discarded, and 100. mu.L of AcN was added and incubated for 5min until the gel became white. Centrifuging at 1000g for 10min, discarding the supernatant, and air drying at room temperature. Add 200. mu.L of 10mM DTT (25 mM NH for 1M DTT)₄HCO₃Diluted), gently shaken and incubated at 56 ℃ for 1h, 100. mu.L of 55mM IAA was quickly added to the gel and incubated at room temperature for 30mins in the dark. With 50mM NH₄HCO₃Adding AcN after rinsing once, repeating for 2-3 times until the colloidal particles become white, and air drying at room temperature. A trypsin solution was added to cover the micelles and incubated at 37 ℃ for 16 h. 1000g centrifugation, supernatant and 50. mu.L 25mM NH₄HCO₃Extracting twice, drying at 80 deg.C, dissolving the sample with 0.1% formic acid water, desalting with C18 column, 20 μ L0.1% formic acid deionized water, and performing mass spectrometryAnalysis and protein identification.

The protein raw data obtained by mass spectrometry is stored in a Proteome distributor^TMAnd (3) performing comparison analysis on Software 2.3 with the obtained securinega suffruticosa protein database to obtain a candidate protein list.

EXAMPLE 83 'and 5' RACE of candidate genes to obtain full-Length genes

The candidate gene Cluster-10344.81450(FsBBE) is likely to be the target gene to be searched. However, the gene has no full length in transcriptome data, and is only 438bp, so that the full-length sequence of the gene needs to be obtained by a RACE method. Primers required for 3 'and 5' RACE were designed based on the existing sequences as described in the materials section. RACE method is shown in clongtech kit. The full-length sequence of the obtained gene (FsBBE) is shown as SEQ ID NO. 1, and the amino acid sequence of the coded oxidase is shown as SEQ ID NO. 2.

Example 9 heterologous expression, protein purification and functional verification of candidate genes

OD was tested by growing about 24h in LB medium containing rifamycin and kanamycin with GV3101 Agrobacterium containing the constructed pGambia 2301::81450-HIS Tag (with BamHI and SalI cleavage sites) vector₆₀₀After centrifugation at 4000g, the cells were washed with buffer (10mM MES,10mM MgCl, 1.0 g)₂And 100. mu.M acetosyringone, pH 5.6) resuspended to OD₆₀₀After 0.6, injecting tobacco, collecting tobacco leaves after 4 days of injection, freezing and grinding, and extracting protein by the same method as that of extracting the total protein of the suffruticosa: 1g fresh leaves were freeze-ground with liquid nitrogen and extracted for 1h on ice using 5mL of buffer A (100mM NaPi, pH 7.4,5mM sodium bisulfite, 5mM dithiothreitol, 1mM EDTA, 10% (v/v) glycerol, 1% (w/v) PVP, 4% (w/v) PVPP) at 4 ℃. Centrifuge at 5,000x g for 30min at 4 ℃. The supernatant was desalted with pD10 columns (GE-Healthcare) and buffer B (10mM Tris-HCl, pH 7.5) was replaced. Then purifying the reconstructed protein of the Cluster-10344.81450 gene HIS-TAG label, adsorbing the total protein by a nickel column, eluting by imidazole with different gradients, verifying by SDS PAGE (see figure 7) of fractions washed by 100mM imidazole, desalting by a pD10 desalting column, replacing with Tris-HCl pH 7.5 buffer solution, and performing enzyme activity test, wherein the test result is shown inFig. 4.

As can be seen from the left graph in FIG. 4, after the FsBBE protein is expressed in tobacco in a heterologous way, the total tobacco leaf protein can also catalyze allocorenine to perform oxidative condensation with L-AA or DHA or isoascorbyl acid to obtain the compound 1 or 2 (see iii, iv and v in the right graph).

Example 10 validation of the catalytic mechanism of oxidase FsBBE

Incubating FsBBE protein purified from tobacco leaves and allosecurinine, taking heat-inactivated protein as a control group, incubating tobacco leaf total protein expressing Cluster-10344.81450 gene and allosecurinine, injecting unloaded pGambia2301 Agrobacterium tumefaciens tobacco leaf total protein as a control group, reacting for 3 hours at 30 ℃, adding equal volume of methanol to terminate the reaction, centrifuging 17000g to take supernatant, injecting 1 mu L of sample, and detecting by Q-TOF. The results are shown in FIG. 4.

Incubating tobacco leaf total protein expressing Cluster-10344.81450 gene with allosecurinine, reacting at 30 ℃ for 3h, adding equal volume of n-butanol, extracting for 3 times, combining n-butanol phases, spin-drying, separating liquid phase, spin-drying separated compound 3 (enamine intermediate) component, dissolving with 40 μ L of methanol, and performing the following experiment: mu.L of Compound 3 solution was added to 50. mu.L of Tris-HCl pH 7.5 buffer as a control (Compound 3); 1 μ L L-AA (100mM in ddH)₂O) adding 50. mu.L Tris-HCl buffer solution with pH 7.5 to the aqueous solution to obtain a control group (L-AA); 1 μ L of Compound 3 solution and 1 μ L L-AA added to 50 μ L Tris-HCl pH 7.5 buffer as experimental group (Compound 3+ L-AA); DHA is analogous to the reaction above, in D₂Experiments in O except that the solution was D₂The rest of the buffer solution prepared by O is the same, after reaction is carried out for 3 hours at 30 ℃, the sample is injected with 1 mu L of filter membrane filtration and Q-TOF detection. The results are shown in FIG. 4.

Example 11 oxidase FsBBE catalyzes the synthesis and structural characterization of another novel compound

When looking for L-AA analogs, only erythorbic acid (isoascorbyl acid) was available, and therefore activity tests were performed with purified FsBBE protein and allocoreurinine and erythorbic acid, and the test results are shown in FIG. 4. The method for separating, purifying and identifying the large-scale enzyme activity of the new compound comprises the following steps: desalting total protein of tobacco leaf expressing Cluster-10344.81450 gene, adding allocoreurinine with final concentration of 1mM and isoascorbic acid with final concentration of 1mM, reacting at 30 deg.C for 3h, adding n-butanol with equal volume, extracting for 3 times, mixing n-butanol phases, spin drying, and separating liquid phase, wherein the liquid phase separation method is the same as that for separating compound 1. Respectively obtaining a compound 2, dissolving in deuterated chloroform and deuterated methanol, and performing nuclear magnetic identification. Both compounds were single crystal tried in different solvents, compound 2 in ethyl acetate: the methanol was slowly evaporated at room temperature in a solvent of 4:1 to obtain crystals, and the absolute configuration was determined by crystal diffraction (see fig. 8). That is, the molecular structural formula of compound 2 is as follows:

the molecular structure of the compound is different from that of the compound 1 in the spatial configuration of 6' -hydroxyl, and the compound is named fluesuffine B.

EXAMPLE 12 two novel Compound physiological Activity assays

The growth inhibitory activity of compound 1(fluesuffine A) and compound 2(fluesuffine B) on tumor cells was tested by the SRB method (Skehan P.et al.1990).

The tumor cell strain is glioma cell SF-268, breast cancer cell MCF-7, liver cancer cell HePG-2, non-small cell lung cancer cell A549 and hepatic astrocyte LX-2.

The experimental method comprises the following steps: respectively dissolving the compound 1(fluesuffine A) and the compound 2(fluesuffine B) in dimethyl sulfoxide (DMSO) to obtain mother liquor with the concentration of 10mmol/L, and then diluting the mother liquor to the required concentration by using an RPMI-1640 culture medium, wherein the positive control medicament is cisplatin. Collecting SF-268, MCF-7, HepG-2, A549 and LX-2 cells in logarithmic growth phase, digesting with pancreatin, staining and counting with trypan blue, detecting cell viability to be more than 95% by trypan blue exclusion experiment, and adjusting cell concentration to 3 × 10 with fresh RPMI-1640 culture medium⁴one/mL, cells were seeded in 96-well plates, 180. mu.L of cell suspension was added to each well, and 3 blank wells were set to zero, at 37 ℃ with 5% CO₂Culturing in an incubator for 24 h. After the cells adhere to the wall, 20 mu L of the mixture with a certain concentration is added into each holeCompound solution, negative control plus 20. mu.L RPMI-1640 medium, with cisplatin as positive control. Placing at 37 ℃ and 5% CO₂After culturing for 72h in an incubator, 50 μ L of 50% cold trichloroacetic acid is added to fix the cells, the cells are placed at 4 ℃ for 1h, washed with distilled water for 5 times, and naturally dried in the air. Then, 100. mu.L/well of SRB solution prepared from 1% glacial acetic acid and having a concentration of 4mg/mL was added, stained at room temperature for 30min, the supernatant was removed, and washed 5 times with 1% glacial acetic acid. Finally adding 200 mu L/hole 10mmol/mL Tris solution for dissolution, measuring the absorbance value (A) at 570nm by a microplate reader, and calculating the inhibition rate of the drug on the cell growth by using the following formula: cell growth inhibition (%) - (1-A)_{Sample set}/A_{Control group})×100％。

The experimental results are as follows: the IC of the compound 1, the compound 2 and the positive contrast medicament cisplatin prepared by the invention on tumor cell strains SF-268, MCF-7, HePG-2, A549 and LX-2₅₀The values are shown in Table 2.

TABLE 2 inhibitory Activity of Compounds 1 and 2 on tumor cell growth: (

n＝3)

The results show that: the compound 1 and the compound 2 have obvious growth inhibition activity on tumor cells, so the implementation of the invention provides candidate compounds for researching and developing new antitumor drugs, and provides scientific basis for developing and utilizing traditional Chinese medicine plant resources.

The biosynthesis of the compound 1 and the compound 2 lays a foundation for analyzing an alkaloid synthesis path in the securinega suffruticosa so as to promote the development and utilization of new natural medicines.

Sequence listing

<110> China academy of sciences molecular plant science remarkable innovation center

<120> Securinega suffruticosa derived oxidase and application thereof

<130> SHPI2010290

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1599

<212> DNA

<213> Flueggea suffruticosa (Pall.) Baill.

<220>

<221> CDS

<222> (1)..(1596)

<400> 1

atg aat cca tta aag cac tct tca tca acg cct tta gtg ttt gtg ttg 48

Met Asn Pro Leu Lys His Ser Ser Ser Thr Pro Leu Val Phe Val Leu

1 5 10 15

ctg act gta tgt tca tgt gca act tca gta aca att cct gag ctg ttc 96

Leu Thr Val Cys Ser Cys Ala Thr Ser Val Thr Ile Pro Glu Leu Phe

20 25 30

ttt caa tgc ctg tcc aat aca acc aca acc agt act agt att ttc aat 144

Phe Gln Cys Leu Ser Asn Thr Thr Thr Thr Ser Thr Ser Ile Phe Asn

35 40 45

gtc cta tac aca cca aga aac acg tcc tac act tcc atc tta gaa tca 192

Val Leu Tyr Thr Pro Arg Asn Thr Ser Tyr Thr Ser Ile Leu Glu Ser

50 55 60

cgc att caa aac ctc agg ttc aat aca act gac acg ccg aaa cct ctg 240

Arg Ile Gln Asn Leu Arg Phe Asn Thr Thr Asp Thr Pro Lys Pro Leu

65 70 75 80

gcc ata gtc aca ccg ctg gat gca tct cac att caa gcc acc atc ata 288

Ala Ile Val Thr Pro Leu Asp Ala Ser His Ile Gln Ala Thr Ile Ile

85 90 95

tgt gcc cgg aaa cac aac ctc caa atc aga atc cga agc ggt ggc cac 336

Cys Ala Arg Lys His Asn Leu Gln Ile Arg Ile Arg Ser Gly Gly His

100 105 110

gac tat gag ggg ttg tct tat gtc tcg ccc ctc cct ttt gtt gtg ctt 384

Asp Tyr Glu Gly Leu Ser Tyr Val Ser Pro Leu Pro Phe Val Val Leu

115 120 125

gat cta atc aat ctt cga aac atc acc gtt gat gta gaa aat aga gtc 432

Asp Leu Ile Asn Leu Arg Asn Ile Thr Val Asp Val Glu Asn Arg Val

130 135 140

gca tgg gtc ggg tgc gga gca aca tta gga gaa ttc tac tat aga att 480

Ala Trp Val Gly Cys Gly Ala Thr Leu Gly Glu Phe Tyr Tyr Arg Ile

145 150 155 160

gca gag aaa act agg acc ctg gca ttc cct gca ggt gct tgt cct act 528

Ala Glu Lys Thr Arg Thr Leu Ala Phe Pro Ala Gly Ala Cys Pro Thr

165 170 175

gta gga gtt ggt ggg cat ttc agt gga ggc gga tac ggg tat ttg ttg 576

Val Gly Val Gly Gly His Phe Ser Gly Gly Gly Tyr Gly Tyr Leu Leu

180 185 190

cgt aaa ttt ggc ctc gca gca gat aac atc ctt gat gca agt tta gtt 624

Arg Lys Phe Gly Leu Ala Ala Asp Asn Ile Leu Asp Ala Ser Leu Val

195 200 205

gat gtg aat ggt aga att ctc gat aga gct tcc atg ggg gaa gat ttg 672

Asp Val Asn Gly Arg Ile Leu Asp Arg Ala Ser Met Gly Glu Asp Leu

210 215 220

ttt tgg gca att aga ggt ggt gga gga aat agt ttc gga gta gtt att 720

Phe Trp Ala Ile Arg Gly Gly Gly Gly Asn Ser Phe Gly Val Val Ile

225 230 235 240

gct tgg aag gtt aat ttg gtt cca gtc cct tcc aca ttg act tct ttc 768

Ala Trp Lys Val Asn Leu Val Pro Val Pro Ser Thr Leu Thr Ser Phe

245 250 255

aaa gtc tca aaa agt ttg gaa cag aat acg atg att cag ctt ctc aac 816

Lys Val Ser Lys Ser Leu Glu Gln Asn Thr Met Ile Gln Leu Leu Asn

260 265 270

aag tgg caa tat gtt gca aat aaa ctt cct gat gaa tta agc atg ttt 864

Lys Trp Gln Tyr Val Ala Asn Lys Leu Pro Asp Glu Leu Ser Met Phe

275 280 285

gct gta gtt tct aaa aaa aac tca aca ata tct gtt aag ttt tat tcc 912

Ala Val Val Ser Lys Lys Asn Ser Thr Ile Ser Val Lys Phe Tyr Ser

290 295 300

ttg tat gta ggt gga att gat agc ctc ctt cca tta atg gaa gaa agg 960

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

305 310 315 320

ttt cct gag ctt ggt tta aaa aga gcg gat tgc aat gag atg agc tgg 1008

Phe Pro Glu Leu Gly Leu Lys Arg Ala Asp Cys Asn Glu Met Ser Trp

325 330 335

ata gag tca gca gta tct ttc gcc ggg tac gca agt aat aca tca ttg 1056

Ile Glu Ser Ala Val Ser Phe Ala Gly Tyr Ala Ser Asn Thr Ser Leu

340 345 350

gat gtt ctc ctc aat cac act aat aat tac gaa att gct agt gga aga 1104

Asp Val Leu Leu Asn His Thr Asn Asn Tyr Glu Ile Ala Ser Gly Arg

355 360 365

ttc aaa ggc aaa tcg gac ttt gtc aaa gag ccc gtg cca gaa gct gca 1152

Phe Lys Gly Lys Ser Asp Phe Val Lys Glu Pro Val Pro Glu Ala Ala

370 375 380

tta gaa ggc tta ttg aaa tgg ctt tca gac aaa gac ata acg aat gca 1200

Leu Glu Gly Leu Leu Lys Trp Leu Ser Asp Lys Asp Ile Thr Asn Ala

385 390 395 400

gcg atc tat atg gtt cca tta gga gga aaa atg ggt gag ata aca gaa 1248

Ala Ile Tyr Met Val Pro Leu Gly Gly Lys Met Gly Glu Ile Thr Glu

405 410 415

aca agc att cca ttc cca cat aga gca ggg aat cta tac ttg ttg gcg 1296

Thr Ser Ile Pro Phe Pro His Arg Ala Gly Asn Leu Tyr Leu Leu Ala

420 425 430

tat tat gtt aaa tgg gag ggg caa gga aca gaa gca gct caa aag ccc 1344

Tyr Tyr Val Lys Trp Glu Gly Gln Gly Thr Glu Ala Ala Gln Lys Pro

435 440 445

cta agt tgg atc aga aag ggt tac aaa tac atg gct ccc tat gtc tcc 1392

Leu Ser Trp Ile Arg Lys Gly Tyr Lys Tyr Met Ala Pro Tyr Val Ser

450 455 460

aaa aat cca aga gaa gca cat ctc aac gac aga gat ctt gat att ggt 1440

Lys Asn Pro Arg Glu Ala His Leu Asn Asp Arg Asp Leu Asp Ile Gly

465 470 475 480

act aac aat atc tca gga aat acc agt tac gaa cag gct agt att tgg 1488

Thr Asn Asn Ile Ser Gly Asn Thr Ser Tyr Glu Gln Ala Ser Ile Trp

485 490 495

gga acc aag tat ttt aaa aat aat ttt gac agg ttg gtt cgg gtg aag 1536

Gly Thr Lys Tyr Phe Lys Asn Asn Phe Asp Arg Leu Val Arg Val Lys

500 505 510

act agt gtt gat cct tca gat ttt ttc aga aat gaa caa agc gtc cct 1584

Thr Ser Val Asp Pro Ser Asp Phe Phe Arg Asn Glu Gln Ser Val Pro

515 520 525

cct ctg tta tct tga 1599

Pro Leu Leu Ser

530

<210> 2

<211> 532

<212> PRT

<213> Flueggea suffruticosa (Pall.) Baill.

<400> 2

Met Asn Pro Leu Lys His Ser Ser Ser Thr Pro Leu Val Phe Val Leu

1 5 10 15

Leu Thr Val Cys Ser Cys Ala Thr Ser Val Thr Ile Pro Glu Leu Phe

20 25 30

Phe Gln Cys Leu Ser Asn Thr Thr Thr Thr Ser Thr Ser Ile Phe Asn

35 40 45

Val Leu Tyr Thr Pro Arg Asn Thr Ser Tyr Thr Ser Ile Leu Glu Ser

50 55 60

Arg Ile Gln Asn Leu Arg Phe Asn Thr Thr Asp Thr Pro Lys Pro Leu

65 70 75 80

Ala Ile Val Thr Pro Leu Asp Ala Ser His Ile Gln Ala Thr Ile Ile

85 90 95

Cys Ala Arg Lys His Asn Leu Gln Ile Arg Ile Arg Ser Gly Gly His

100 105 110

Asp Tyr Glu Gly Leu Ser Tyr Val Ser Pro Leu Pro Phe Val Val Leu

115 120 125

Asp Leu Ile Asn Leu Arg Asn Ile Thr Val Asp Val Glu Asn Arg Val

130 135 140

Ala Trp Val Gly Cys Gly Ala Thr Leu Gly Glu Phe Tyr Tyr Arg Ile

145 150 155 160

Ala Glu Lys Thr Arg Thr Leu Ala Phe Pro Ala Gly Ala Cys Pro Thr

165 170 175

Val Gly Val Gly Gly His Phe Ser Gly Gly Gly Tyr Gly Tyr Leu Leu

180 185 190

Arg Lys Phe Gly Leu Ala Ala Asp Asn Ile Leu Asp Ala Ser Leu Val

195 200 205

Asp Val Asn Gly Arg Ile Leu Asp Arg Ala Ser Met Gly Glu Asp Leu

210 215 220

Phe Trp Ala Ile Arg Gly Gly Gly Gly Asn Ser Phe Gly Val Val Ile

225 230 235 240

Ala Trp Lys Val Asn Leu Val Pro Val Pro Ser Thr Leu Thr Ser Phe

245 250 255

Lys Val Ser Lys Ser Leu Glu Gln Asn Thr Met Ile Gln Leu Leu Asn

260 265 270

Lys Trp Gln Tyr Val Ala Asn Lys Leu Pro Asp Glu Leu Ser Met Phe

275 280 285

Ala Val Val Ser Lys Lys Asn Ser Thr Ile Ser Val Lys Phe Tyr Ser

290 295 300

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

305 310 315 320

Phe Pro Glu Leu Gly Leu Lys Arg Ala Asp Cys Asn Glu Met Ser Trp

325 330 335

Ile Glu Ser Ala Val Ser Phe Ala Gly Tyr Ala Ser Asn Thr Ser Leu

340 345 350

Asp Val Leu Leu Asn His Thr Asn Asn Tyr Glu Ile Ala Ser Gly Arg

355 360 365

Phe Lys Gly Lys Ser Asp Phe Val Lys Glu Pro Val Pro Glu Ala Ala

370 375 380

Leu Glu Gly Leu Leu Lys Trp Leu Ser Asp Lys Asp Ile Thr Asn Ala

385 390 395 400

Ala Ile Tyr Met Val Pro Leu Gly Gly Lys Met Gly Glu Ile Thr Glu

405 410 415

Thr Ser Ile Pro Phe Pro His Arg Ala Gly Asn Leu Tyr Leu Leu Ala

420 425 430

Tyr Tyr Val Lys Trp Glu Gly Gln Gly Thr Glu Ala Ala Gln Lys Pro

435 440 445

Leu Ser Trp Ile Arg Lys Gly Tyr Lys Tyr Met Ala Pro Tyr Val Ser

450 455 460

Lys Asn Pro Arg Glu Ala His Leu Asn Asp Arg Asp Leu Asp Ile Gly

465 470 475 480

Thr Asn Asn Ile Ser Gly Asn Thr Ser Tyr Glu Gln Ala Ser Ile Trp

485 490 495

Gly Thr Lys Tyr Phe Lys Asn Asn Phe Asp Arg Leu Val Arg Val Lys

500 505 510

Thr Ser Val Asp Pro Ser Asp Phe Phe Arg Asn Glu Gln Ser Val Pro

515 520 525

Pro Leu Leu Ser

530

Claims (10)

1. An isolated polypeptide selected from the group consisting of:

(a) a polypeptide having the amino acid sequence of SEQ ID NO 2;

(b) 2 by substitution, deletion or addition of one or more amino acid residues, and has (a) polypeptide function;

(c) a polypeptide derived from (a) having a homology of 95% or more, preferably 98% or more, more preferably 99% or more with the polypeptide sequence defined in (a) and having the function of the polypeptide of (a); or

(d) A derivative polypeptide of the polypeptide sequence in (a) or (b) or (c) is contained in the sequence.

2. An isolated polynucleotide selected from the group consisting of:

(A) a polynucleotide encoding the polypeptide of claim 1;

(B) a polynucleotide encoding a polypeptide having the amino acid sequence shown in SEQ ID NO. 2;

(C) the polynucleotide with the nucleotide sequence shown as SEQ ID NO. 1;

(D) polynucleotide whose nucleotide sequence has homology of 95% or more, preferably 98% or more, more preferably 99% or more with the nucleotide sequence shown in SEQ ID NO. 1;

(E) a nucleotide sequence complementary to the nucleotide sequence of any one of (A) to (D).

3. The polynucleotide of claim 2, which is SEQ ID No. 1.

4. A vector comprising the polynucleotide of claim 2 or 3.

5. A microorganism transformed with the vector of claim 4.

6. The microorganism according to claim 5, characterized in that it is E.coli BL21(DE 3).

7. The polypeptide according to claim 1 or the use of the microorganism according to claim 5 or 6 for preparing the securinega suffruticosa alkaloid flueffine A shown in formula 1:

8. the use according to claim 7, wherein the securinega suffruticosa alkaloid fluensuine A is prepared by taking L-ascorbic acid or dehydroascorbic acid and securinega suffruticosa alkali as substrate raw materials and performing an oxidative condensation reaction under the catalysis of the polypeptide according to claim 1.

9. Use of the polypeptide of claim 1, or the microorganism of claim 5 or 6, for the preparation of a compound of formula 2:

10. the use according to claim 9, wherein the preparation is carried out by oxidation-condensation reaction of raw materials including erythorbic acid and securinine as substrates under the catalysis of the polypeptide according to claim 1.

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