CN111662891B - Piericidin glycosyltransferase sbmGT1 and application thereof - Google Patents

Abstract

The invention provides a piericidin glycosyltransferase sbmGT 1; also provides the amino acid sequence and the nucleotide sequence of the glycosyltransferase; and provides a cloning and expression method of the saccharification transferase. The glycosyltransferase sbmGT1 is responsible for the glycosylation modification step of the piericidin in an organism and has the capacity of directionally improving the yield of the glycosylated piericidin; on one hand, the method fills up the technical blank of glycosylation modification after biosynthesis of the piericins compounds in the prior art, and on the other hand, the method greatly enhances the inhibition activity of the piericins compounds on tumor cell proliferation; therefore, the method has important economic value and has more important social significance. Moreover, when the nucleotide sequence of glycosyltransferase sbmGT1 is the preferable nucleotide sequence, the yield of compound 10-glucopericidin A with the best activity of inhibiting tumor cell strain proliferation is obviously improved, and the method has important social significance and can generate great economic benefit.

Description

Piericidin glycosyltransferase sbmGT1 and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering and biological pharmacy, and particularly relates to glycosyltransferase and application thereof in piericidin glycosylation modification.

Background

Piericidins (Piericidins) are alpha-pyridone antibiotics of microbial origin, and more than 60 natural products of Piericidins have been found, all produced by land-and marine-derived actinomycetes. The piericins have reported insecticidal and antibacterial activities, and also have inhibitory activities on part of tumor cells. The invention patent application 201610965145.4 discloses an application of a Piericidin compound Piericidin A in preparing anti-renal cancer drugs. Researchers find that Piericidin A has different-strength inhibition effects on three human renal cancer cell lines, particularly has the strongest inhibition activity on human adrenal gland cancer cell ACHN, so that the Piericidin A can be used as a Bcl-2 inhibitor for development of anti-renal cancer drugs.

Nobutaka Takahashi et al (Journal of the American Chemical Society,1965,87(9): 2066) -2068) isolated in Streptomyces mobaraensis to give piericidin A1 compound; the compound has attracted long-term attention and research as a mitochondrial respiration inhibitor. A series of piericins compounds are separated from Streptomyces sp.KIB-H1083 by Ning-Ning Shang et al (The Journal of Antibiotics,2018,71(7):672-676), and activity experiments prove that 10-glucoptericidin A1 has good inhibitory activity on HL-60, SMMC-772, A-549 and MCF-7 cell strains, while piericidin A1 has no inhibitory activity. In addition, researchers have also conducted a series of studies on the biosynthesis of piericins. Qian Liu et al (Chemistry & Biology,2012,19 (2243-253)) reported the Piericidin A1 biosynthetic gene cluster and resolved the α -pyridine ring formation mechanism Yaolong Chen et al (Organic Letters,2014,16(3):736-739) reported the hydroxylation and post-methylation modification processes in the Piericidin A1 biosynthetic pathway.

Glycosylation modification is a common method used in the biosynthesis of natural products. Glycosylation modification can reduce the toxicity of compounds, and is a self-protection mechanism of strains. Meanwhile, researches find that glycosylation modification can improve the water solubility of the compound, improve the bioavailability of the compound and reduce toxic and side effects. Therefore, the preparation of the glycosylation modified compound has important application value. The invention patent ZL201410620716.1 discloses a breviscapine glycosyl transferase, a preparation method and application thereof; the invention patent ZL201610160405.0 discloses hawthorn fruit cyanidin-3-hydroxyl glycosyl transferase and a coding gene and application thereof; the invention patent 201310283702.0 discloses a glucosyltransferase and application thereof. According to the current literature report, no relevant record about the piericidin glycosyltransferase is found; therefore, the modification pathway of the compound of the piericins after biosynthesis is not complete. Therefore, most of the currently reported glycosylated piericins are natural, and the biggest problem is that the yield cannot be effectively controlled, so that the technical progress and the industrial application are severely restricted.

Disclosure of Invention

In order to solve the technical blank of glycosylation modification after biosynthesis of a piericidin compound in the prior art, the invention provides piericidin glycosyltransferase (an amino acid sequence is derived from marine bacillus B.methylotrophicus B-9987) which is named as glycosyltransferase sbmGT 1. The glycosyltransferase sbmGT1 is responsible for the glycosylation modification step of the piericidin in organisms and has the capacity of directionally improving the yield of glycosylated piericidin.

The technical scheme of the invention is as follows:

the piericidin glycosyltransferase sbmGT1 can effectively catalyze the spiicidin A1 and UDP-D-Glu to carry out glycosylation reaction; the amino acid sequence of the glycosyltransferase is selected from the following (1), (2) or (3):

(1) as shown in SEQ ID NO: 3;

(2) as shown in SEQ ID NO: 3 is subjected to substitution, deletion or addition of one or more amino acids and has an amino acid sequence which catalyzes the glycosylation reaction of piericidin A1 and UDP-D-Glu;

(3) and SEQ ID NO: 3, and the expressed protein has an amino acid sequence for catalyzing the glycosylation reaction of the pieicidin A1 and UDP-D-Glu.

The nucleotide sequence for coding the piericidin glycosyltransferase sbmGT1 is selected from the following (1), (2), (3) or (4):

(1) a nucleotide sequence shown as SEQ ID NO. 2;

(2) a nucleotide sequence which is different from the nucleotide sequence shown in SEQ ID NO. 2 but encodes the amino acid sequence shown in SEQ ID NO. 2;

(3) the homology of the expression protein and a nucleotide sequence shown in SEQ ID NO. 2 is more than or equal to 85 percent, and the expressed protein has the nucleotide sequence for catalyzing the glycosylation reaction of piericidin A1 and UDP-D-Glu;

(4) a nucleotide sequence complementary to the nucleotide sequence of any one of (1), (2) or (3).

Contains the nucleotide sequence for coding the piericidin glycosyltransferase sbmGT 1. The expression vector is a vector suitable for expression in Escherichia coli.

Preferably, the expression vector is an integrative vector; the integrated vector is a vector suitable for high expression in actinomycetes.

A piericidin glycosyltransferase sbmGT1 is obtained by transforming an Escherichia coli expression system for expression.

The cloning expression method of the piericidin glycosyltransferase sbmGT1 comprises the following steps: cloning the nucleotide sequence coding the glycosyltransferase sbmGT1 into an expression vector to construct an expression vector; then transferring the expression vector into an expression system for expression; finally purifying to obtain the piericidin glycosyltransferase sbmGT 1.

The piericidin glycosyltransferase sbmGT1 is applied to the preparation of glycosylation modified compounds. Wherein, the compound is a piericidin compound. Specifically, the piericidin compound is piericidin A1, and the glycosylation modification is to perform glycosylation modification on the 10-position hydroxyl and/or the 4' -position hydroxyl of the piericidin A1 compound through enzyme catalysis reaction.

The application is that the glycosyl of a glycosyl donor is transferred and combined to the piericidin compound by the piericidin glycosyl transferase sbmGT1, and UDP-D-Glu is preferably used as the glycosyl donor.

The reaction system (100. mu.L) of the enzyme catalytic reaction: 2.5M Tris-HCl buffer (pH8.0) 1. mu.L; 0.1mM MgCl ₂1 μ L; 15mM Compound 1: 2. mu.L; 5 mu L of 20mM UDP-D-Glu; 5 μ L of 0.2mM sGT1 protein; ddH₂O86 μ L. Placing the mixture in a water bath at 30 ℃ for reaction for 1 h.

The application specifically comprises the following steps: (1) cloning the nucleotide sequence coding the glycosyltransferase sbmGT1 into an integrative vector to construct an integrative expression vector; (2) transferring the recombinant expression vector into streptomyces S.youssoufield OUC6819 for high expression; (3) and purifying to obtain the glycosylated piericins compound.

Preferably, when the nucleotide sequence of the glycosyltransferase sbmGT1 is the nucleotide sequence shown in SEQ ID NO. 2, the glycosylation modification of the piericidin compound is realized, and the yield (accounting for 82.50 wt%) of 10-glucopericidin A (compound 2) with the best tumor cell strain proliferation inhibition activity is remarkably improved, so that the important social significance is realized, and great economic benefit is generated.

The invention has the beneficial effects that:

(1) the invention provides a glycosyltransferase sbmGT1 for the first time, wherein the glycosyltransferase is used for glycosylation modification of piericidin; the application of the derivative in catalyzing the glycosylation reaction of the piericidin A1 and UDP-D-Glu is disclosed, the technical blank of glycosylation modification after biosynthesis of the piericidin compounds in the prior art is filled, and the derivative has important significance.

(2) The invention provides important technical support for glycosylation modification after biosynthesis of the piericins compound, realizes yield optimization and large-scale fermentation preparation by molecular genetic manipulation means, and lays a solid foundation for marketization of the piericins compound.

(3) The glycosyltransferase sbmGT1 is adopted to carry out glycosylation modification on the piericidin compound, so that the yield of glycosylated piericidin is directionally improved, and the inhibitory activity of the glycosylated piericidin on tumor cell proliferation is greatly enhanced; therefore, the method has important economic value and has more important social significance.

Drawings

FIG. 1 is a diagram showing the structures of compounds 2 to 7 obtained by the present invention and Piericidin A1;

FIG. 2 is an ultraviolet absorption spectrum of the piericins compound 1-7 of the present invention;

FIG. 3 is an SDS-PAGE analysis of in vitro expression of sbmGT1 protein according to the invention;

FIG. 4 shows the HPLC detection results of the in vitro enzymatic reaction of sbmGT1 protein of the present invention;

FIG. 5 shows the HPLC analysis result of the fermentation product of the recombinant strain I;

FIG. 6 is a Mass Spectrum (MS) of piericidin A1(1) in accordance with the present invention;

FIG. 7 is a Mass Spectrometry (MS) spectrum of Compound 2 of the present invention;

FIG. 8 is a Mass Spectrometry (MS) spectrum of Compound 3 of the present invention;

FIG. 9 is a Mass Spectrometry (MS) spectrum of Compound 4 of the present invention;

FIG. 10 is a Mass Spectrometry (MS) spectrum of Compound 5 of the present invention;

FIG. 11 is a Mass Spectrometry (MS) spectrum of Compound 6 of the present invention;

FIG. 12 is a Mass Spectrometry (MS) spectrum of Compound 7 of the present invention.

Detailed Description

The present invention will be further described with reference to the following examples.

Example 1: cloning expression of glycosyltransferase gene sbmGT1 and in vitro enzyme catalytic reaction

1. Glycosyltransferase codon optimization

The nucleotide sequence of the glycosyltransferase gene bmmGT1 is shown in SEQ ID NO. 1, the codon optimization is based on the codon preference of Streptomyces coelicolor M145, and the specific codon preference is as follows: alanine ala (gcc); arginine arg (cgc); asparagine asn (aac); aspartic acid Asp (GAC); cysteine cys (ugc); glutamic acid is selected from Gln (CAG); glutamic acid glu (gag); glycine gly (ggc); histidine his (cac); isoleucine ile (auc); leucine leu (cug); lysine lys (aag); methionine met (aug); phenylalanine phe (uuc); proline pro (ccg); serine ser (ucc); threonine thr (acc); tryptophan trp (ugg); tyrosine tyr (uac); valine val (guc); stop codon (TAA). The modified nucleotide fragment was named sbmGT1 and was entrusted to the company for gene synthesis, and the nucleotide sequence was shown in SEQ ID NO. 2.

2. Construction of expression vectors

Designing a primer pair:

P1：5’-ggaattccatatgcgcaagacccacatcgc-3’/2：5’-ccgctcgaggttctcgacggcggcgg-3'. PCR was performed using the artificially synthesized DNA fragment as a template. Wherein the primer pair P1/P2 is used to amplify codon-optimized glycosyltransferase genes, the restriction sites for NedI (catatg), XhoI (ctcgag) are underlined.

And (3) PCR reaction system:

primer pairs P1 and P2, 5. mu.L each (50pmol), template 5. mu.L, 10 × Reaction Buffer 10. mu.L, 2.5mM dNTP 10. mu.L, 25mM MgCl ₂6 μ L, 1 μ L (5U/. mu.L) of Taq DNA Polymerase, plus ddH₂O to 100. mu.L.

PCR conditions were as follows:

under the condition of promoter amplification, denaturation is carried out for 5min at 95 ℃; 30s at 95 ℃, 30s at 60 ℃ and 30s at 72 ℃ for 28 cycles; 5min at 72 ℃; functional gene amplification condition, denaturation at 95 ℃ for 5 min; 30s at 95 ℃, 30s at 65 ℃, 1min at 72 ℃ for 30s, and 28 cycles; 5min at 72 ℃. Functional genes and a protein expression vector pET28a are connected by T4 ligase, escherichia coli DH5 alpha competent cells are transformed, positive clones are selected and subjected to sequence determination, and a recombinant vector pET28a-sbmGT1 is constructed. Then introducing into Escherichia coli BL21(DE3) cell, inducing expression of target protein with isopropyl thiogalactoside (IPTG), ultrasonic disrupting thallus, affinity purifying with Ni column, ultrafiltering, and concentrating to obtain high-purity enzyme extract. The molecular weight and purity of the obtained target protein were checked by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the results are shown in FIG. 3, wherein the protein has a molecular weight of 47.8kDa and a good protein purity. Measuring the concentration of enzyme extract by BIOFORD method, adding glycerol 50% of the total volume of the enzyme extract and DTT with final molar concentration of 0.01mmol/L, and storing at-20 deg.C.

3. In vitro enzyme activity detection

In vitro enzyme activity reaction system (100 mu L)

2.5M Tris-HCl buffer (pH8.0): 1. mu.L

0.1mM MgCl₂:1μL

15mM piericidin A1:2μL

20mM UDP-Glu:5μL

5 μ L of 0.2mM sGT1 protein

ddH₂O:86μL

The reaction conditions are 30 ℃,60min, after the reaction is finished, 100 mu L of methanol is added, vortex oscillation is carried out for 5min, centrifugation is carried out for 20min at 13,000rpm, the precipitate is discarded, and the supernatant is obtained for HPLC detection.

And (4) HPLC detection: reversed phase C18 column (specification: 150X 4.6mm, 5 μm) was used; the column temperature is 30 ℃; elution conditions: and (3) balancing for 0-5 min: 80% of phase A (ddH)₂O + 0.1% formic acid) and 20% B phase (acetonitrile + 0.1% formic acid); linear elution for 5-45min, 80-0% of phase A and 20-100% of phase B; isocratic elution for 45-50 min: 0% of phase A and 100% of phase B; the detection wavelength is 260 nm; the flow rate was 1 mL/min. From the HPLC spectrum (FIG. 4, ii), a new absorption peak consistent with the substrate UV absorption spectrum (FIG. 2) appeared after the reaction, indicating the occurrence of the enzymatic reaction. By comparison with the standard substance, it was confirmed that compound 2 was mainly produced in the reaction solution; and sbmGT1 protein can completely convert piericidin A1 into glycosylation product, the main product is compound 2.

Example 2: construction of recombinant Strain I

Designing a primer pair: p3: 5'-atgcgcaagacccacatcgc-3'/P4: 5' -gctctagattagttctcgacggcggcgg-3’，P5：5’-ggaattcccgtcgcggaaagctggc-3'/P6: 5'-gaaccgatctcctcgttggtg-3', respectively; PCR was performed using the DNA fragment artificially synthesized in example 1 as a template. Wherein the primer pair P3/P4 is used to amplify codon optimized glycosyltransferase gene, and the primer pair P5/P6 is used to amplify glyceraldehyde triphosphate dehydrogenase gene promoter P_gapDHThe restriction sites for XbaI (tctaga), EcoRI (gaattc) are underlined.

And (3) PCR reaction system:

primer pairs P3 and P4(P5 and P6) were each 5. mu.L (50pmol), template 5. mu.L, 10 × Reaction Buffer 10. mu.L, 2.5mM dNTP 10. mu.L, 25mM MgCl ₂6 μ L, 1 μ L (5U/. mu.L) of Taq DNA Polymerase, plus ddH₂O to 100. mu.L.

PCR conditions were as follows:

under the condition of promoter amplification, denaturation is carried out for 5min at 95 ℃; 30s at 95 ℃, 30s at 60 ℃ and 30s at 72 ℃ for 28 cycles; 5min at 72 ℃; functional gene amplification condition, denaturation at 95 ℃ for 5 min; 30s at 95 ℃, 30s at 65 ℃, 1min at 72 ℃ for 30s, and 28 cycles; 5min at 72 ℃. The promoter and the functional gene are connected with an integrative vector pSET152 by T4 ligase, escherichia coli DH5 alpha competent cells are transformed, positive clones are selected and subjected to sequence determination, and a recombinant vector pSET152-pG-sbmGT1 is constructed.

The constructed recombinant vector pSET152-pG-sbmGT1 is introduced into the streptomyces S.youssoufield OUC6819 strain to obtain a recombinant strain I.

Example 3: production of glycosylated piericidin by recombinant strain I

(1) Culturing spores: inoculating appropriate amount of recombinant strain I to R according to conventional method for culturing microorganism₂YE solid slant culture medium is placed in a 30 ℃ constant temperature incubator for 3-4 days.

R₂YE culture medium: sucrose (103 g, K)₂SO₄ 0.25g，MgCl₂10.12g, 10g of glucose, 0.1g of casein hydrolysate, 5g of yeast extract, dissolved in water, 22g of agar powder, the volume is 800mL, and the mixture is sterilized at 115 ℃ for 30 minutes. Sterilizing, adding 0.5% KH₂PO₄ 10mL，3.68％CaCl₂80mL, 15mL of 20% L-proline, 100mL of 5.73% TES buffer (pH 7.2), 5mL of 1M sodium hydroxide and 2mL of trace salt solution, mixing, pouring the culture medium into a culture dish with the diameter of 90mm, and subpackaging by 30 mL/plate.

(2) Fermentation culture

And taking a proper amount of spores of the recombinant strain I which is cultured on a flat plate for 3-4 days, inoculating the spores into a 250mL conical flask filled with 50mL of culture solution, placing the conical flask in a constant temperature shaking table at 30 ℃, and culturing for 7 days at the rotating speed of 220rpm to obtain mycelium and fermentation liquor. Wherein, the culture medium comprises the following components: soluble starch 100g, KH₂PO₄ 5g，MgSO₄·7H₂O5 g, glucose 200g, yeast extract 100g, corn steep liquor 40g, beef extract 30g, CaCO₃20g of sea salt and 300g of tap water are dissolved to a constant volume of 1L, and the pH value is adjusted to 7.2.

(3) Obtaining and HPLC detecting of fermentation product

The fermentation broth and mycelia were centrifuged at 7500 rpm. Soaking the mycelia with methanol overnight, evaporating methanol under reduced pressure, mixing with the fermentation broth, extracting the mixed fermentation broth with ethyl acetate for three times, mixing ethyl acetate phases, and concentrating under reduced pressureAnd carrying out HPLC detection on the obtained fermentation sample. And (4) HPLC detection: reversed phase C18 column (specification: 150X 4.6mm, 5 μm) was used; the column temperature is 30 ℃; elution conditions: and (3) balancing for 0-5 min: 80% of phase A (ddH)₂O + 0.1% formic acid) and 20% B phase (acetonitrile + 0.1% formic acid); linear elution for 5-45min, 80-0% of phase A and 20-100% of phase B; isocratic elution for 45-50 min: 0% of phase A and 100% of phase B; the detection wavelength is 260 nm; the flow rate was 1 mL/min.

From the HPLC profile (FIG. 5, ii), a new absorption peak consistent with the Piericidin A1 UV absorption spectrum (FIG. 2) appeared in recombinant strain I, indicating that glycosylation modification reactions occurred in vivo. From the analysis of HPLC results, the main product is compound 2 (10-glucopericidin A), and a series of trace amounts of other glycosylated triptycepin compounds are generated. This shows that the glycosyltransferase gene sbmGT1 described in the present application can not only realize the glycosylation modification of the piericidin compound, but also greatly improve the yield of the compound 2 (10-glucopericidin A), realize the directional production of 10-glucopericidin A, and reduce the workload of subsequent purification and separation, thereby further reducing the production cost and simplifying the production steps, and having important significance in industry.

Example 4: preparation of glycosylated piericins compound 2-7

1. Fermentative production was as shown in example 3, scale-up to 20L.

2. Obtaining extract

The fermentation broth and mycelia were centrifuged at 7500 rpm. Soaking the mycelia with methanol overnight, evaporating methanol under reduced pressure, mixing with the fermentation broth, extracting the mixed fermentation broth with equal amount of ethyl acetate for three times, mixing ethyl acetate phases, and concentrating under reduced pressure to obtain crude extract.

3. Separation and purification of Compound

The extract is firstly extracted twice by liquid-liquid extraction with equal volume of normal hexane-95% methanol, and the solvent is removed under reduced pressure. Then, the 95% methanol phase is subjected to reverse phase silica gel column chromatography, and gradient elution is carried out by taking methanol-water as a solvent, and the methanol-water is divided into 14 fractions. Fr-7 (methanol-water 65: 35 eluate, 142.57mg), Fr-8 (methanol-water 70: 30 eluate, 204.28mg), Fr-9 (methanol-water 75: 25 eluate, 351.10mg) and Fr-11 (methanol-water 85: 15 eluate, 80.87 mg). Semi-preparative reverse phase high performance liquid chromatography (acetonitrile: water ═ 48: 52, 60min) afforded compound 7(5.02mg, 5.15 wt%), compound 6(3.08mg, 3.16 wt%), compound 5(3.35mg, 3.43 wt%), compound 4(2.32mg, 2.38 wt%) and compound 3(6.65mg, 6.82 wt%); compound 2(80.49mg, 82.50 wt%) was obtained by semipreparative reverse phase high performance liquid chromatography (acetonitrile: water: 58: 42, 60min), and piericidin a1 (compound 1) (10.74mg) was obtained by semipreparative reverse phase high performance liquid chromatography (acetonitrile: water: 75: 25, 60 min).

In conclusion, the content of compound 2 in the piericidin compounds obtained by using the glycosyltransferase gene sbmGT1 described in the present application is as high as 82.50%, which further corroborates the conclusion obtained in example 2 that the targeted production of 10-glucopericidin A (compound 2) can be achieved by using the artificially synthesized glycosyltransferase gene sbmGT 1.

Example 5: characterization of Compounds 2-7 and Piericidin A1 (Compound 1)

Compound 2 is a pale yellow amorphous solid, formula C₃₁H₄₇NO₉HR-ESIMS (FIG. 7) M/z 578.3309[ M + H [ ]]⁺. Wherein,¹h and¹³the C-NMR data are shown in Table 1.

TABLE 1 preparation of Compound 2¹H and¹³c NMR data (500 and 150MHz, in CDCl)₃)

The signal assignments in table 1 are based on H-H COSY, HMQC and HMBC mapping results. The multiplicity of carbon signals is represented by s (singlet), d (doublet), t (triplet) and m (multiplet), respectively.

Compound 3 is a pale yellow amorphous solid, formula C₃₇H₅₇NO₁₄HR-ESIMS (FIG. 8) M/z 740.3844[ M + H [ ]]⁺。

Compound 4 is a pale yellow amorphous solid with the molecular formula C₃₇H₅₇NO₁₄HR-ESIMS (FIG. 9)/z 740.3848[ M + H ]]⁺。

Compound 5 is a pale yellow amorphous solid, formula C₃₇H₅₇NO₁₄HR-ESIMS (FIG. 10) M/z 740.3840[ M + H [ ]]⁺。

Compound 6 is a pale yellow amorphous solid with the molecular formula C₃₆H₅₅NO₁₃HR-ESIMS (FIG. 11) M/z 710.3738[ M + H [ ]]⁺。

Compound 7 is a pale yellow amorphous solid, formula C₃₆H₅₅NO₁₃HR-ESIMS (FIG. 12) M/z 710.3737[ M + H [ ]]⁺。

Compound 1 is a pale yellow amorphous solid, formula C₂₅H₃₇NO₄HR-ESIMS (FIG. 6) M/z 416.2796[ M + H [ ]]⁺。

Example 6: testing of antitumor Properties of Compounds 1-7

(1) Experimental sample and experimental method

The test samples are pure products of the compounds 1 to 7 separated and purified in the example 4. Accurately weighing a proper amount of sample, and preparing a solution with required concentration by using DMSO (dimethyl sulfoxide) for measuring the activity.

The cell line and the subculture of the cells adopt human colon cancer cells HCT-116 and HT-29, human non-small cell lung cancer cells A549 and human malignant melanoma cells A375, and the cells are subcultured in a culture box which is filled with 5A, 5A, F-12K and DMEM culture medium containing 10% FBS, 100U/mL penicillin and 100 mu g/mL streptomycin at 37 ℃. The liquid is changed every two days, after the cells are converged, the cells are digested by 0.05 percent pancreatin-EDTA and are passaged, and the cells are kept in a good tested logarithmic growth phase.

Cell proliferation assay the inhibition rate of the samples on tumor cells: 5000 tested tumor cells/well are inoculated to a 96-well plate, and after 24 hours of culture, tested samples with different concentrations are added. Positive control doxorubicin hydrochloride (final concentration of 1. mu.M), blank control group was added with equal volume of culture medium, 4 duplicate wells for each concentration. After 72h of drug action, 50% (m/v) cold trichloroacetic acid (TCA) was added to each well to immobilize the cells, which were stained with SRB and then measured for OD540 nm on a microplate reader.

The inhibition rate of tumor cell growth was calculated according to the following formula:

inhibition (%) [ (OD540 blank-OD 540 test set)/OD 540 blank ] × 100.

(2) Results of the experiment

Note: IC (integrated circuit)₅₀The (μ M) is the amount of compound that inhibits tumor cell growth by half of normal levels.

(3) Conclusion

As can be seen from the above table, IC of Compound 2 against four tumor cell lines₅₀0.16. mu.M, 0.97. mu.M, 2.24. mu.M and 0.80. mu.M, respectively, and IC of Compound 5 against four tumor cell lines₅₀Respectively at 0.27. mu.M, 2.34. mu.M, 1.12. mu.M and 0.74. mu.M, indicating that the compounds 2 and 5 have good proliferation inhibition activity on the four tumor cell lines.

Compound 4, compound 6 and compound 7 still have certain proliferation inhibitory activity on four tumor cell lines, but are slightly lower than compound 2 and compound 5. While compound 3 and piracidin A1 (compound 1) before glycosylation modification did not have proliferation inhibitory activity against the above four tumor cell lines. This indicates that the glycosylated piericins obtained by using the glycosyltransferase sbmGT1 all had enhanced tumor cell growth inhibitory activity compared to that before glycosylation modification 1, although they had different tumor cell growth inhibitory activity. Among them, compound 2, which yielded the highest amount (82.50 wt%), had the highest proliferation inhibitory activity against four groups of tumor cells. This means that the glycosyltransferase sbmGT1 described herein not only achieves glycosylation modification of the piericidin compound, but also selectively achieves high yield of the compound 2 with the best tumor suppression effect, which is of decisive significance for the subsequent industrialization process.

Sequence listing

<110> China oceanic university

<120> piericidin glycosyltransferase sbmGT1 and application

<160> 3

<170> SIPOSequenceListing 1.0

<210> 3

<211> 1185

<212> DNA

<213> Bacillus methylotrophicus (B. methylotrophicus B-9987)

<400> 3

atgagaaaaa cacatatcgc cattattaat gtggccgctc acggtcatgt caatcccacg 60

ctgcccgtag ccgaggaact tgtcaatcgc gggtataaag tgacattcgc tacgactgag 120

gaattcgaag cttccgttgc caaaacgggc gccattcccg ttttataccg cacaagcata 180

aaggcggacc cggagacgat aaaagaacgg gtaaataaaa atgacgcgtt tgtgatgttt 240

ctggaagaag cggttgaagt gcttccgcag ctggaagagc tgtacaaaga cgaccttcct 300

gacatcgtcc tgtttgattt tttagctctt gccggaagat tgtttgcgga taatggcggc 360

atcccggccg tgaagttctg cccgagctat gcaatgaatg agtattttca attaggaaga 420

gacgaagaaa cactggaagc ggccaaacag gcgatagaag aataccagga gcagattgaa 480

aatgaacaat tgaaacggat gacaatggaa gaatttttta tgcctgaaaa attgaatatt 540

gtctttatgc caagggcctt tcagccgaaa caggaaacct ttgatgaacg attctgtttt 600

gtgggtccgt ctcttggaga acgcacaaat acgggaagtc tggaaattga tgcagccgat 660

gaccgtccgc ttatgctcat ctcactggga acggcgttta acgcctggcc tgaattttac 720

cggatgtgta ttgacgcgtt tcgggactcg gactggcggg ttattatgtc agtcggcaca 780

acaattgatc cggaaagttt ttcggatgtc cccgatcact ttatcatccg ccagcacgtt 840

cctcagcttg atgtgctgga gaaagcaaag ctgttcgtat ctcacggcgg gatgaacagt 900

acaatggagg cgatgaatgc gggtgtcccg ctcgttgtcg ttccgcaaat gcacgaacaa 960

gaggttaccg caaagcgtgt ggatgaattg gggcttggcg tgcatctgcc gccgaaagaa 1020

gtcactgtgg cccgtttgca aaaagacgtt caaaacgtat acggcgataa aaacatcctc 1080

agccgcgtga aaagcatgca gcagaaaata gaagaaaccg gcggccccaa acaggctgtt 1140

tgcgccattg aagaattttt gaaaacggcg gccgtcgaaa attga 1185

<210> 2

<211> 1185

<212> DNA

<213> Bacillus methylotrophicus (B. methylotrophicus B-9987)

<400> 2

atgcgcaaga cccacatcgc catcatcaac gtcgccgccc acggccacgt caacccgacc 60

ctgccggtcg ccgaggagct ggtcaaccgc ggctacaagg tcaccttcgc caccaccgag 120

gagttcgagg cctccgtcgc caagaccggc gccatcccgg tcctgtaccg cacctccatc 180

aaggccgacc cggagaccat caaggagcgc gtcaacaaga acgacgcctt cgtcatgttc 240

ctggaggagg ccgtcgaggt cctgccgcag ctggaggagc tgtacaagga cgacctgccg 300

gacatcgtcc tgttcgactt cctggccctg gccggccgcc tgttcgccga caacggcggc 360

atcccggccg tcaagttctg cccgtcctac gccatgaacg agtacttcca gctgggccgc 420

gacgaggaga ccctggaggc cgccaagcag gccatcgagg agtaccagga gcagatcgag 480

aacgagcagc tgaagcgcat gaccatggag gagttcttca tgccggagaa gctgaacatc 540

gtcttcatgc cgcgcgcctt ccagccgaag caggagacct tcgacgagcg cttctgcttc 600

gtcggcccgt ccctgggcga gcgcaccaac accggctccc tggagatcga cgccgccgac 660

gaccgcccgc tgatgctgat ctccctgggc accgccttca acgcctggcc ggagttctac 720

cgcatgtgca tcgacgcctt ccgcgactcc gactggcgcg tcatcatgtc cgtcggcacc 780

accatcgacc cggagtcctt ctccgacgtc ccggaccact tcatcatccg ccagcacgtc 840

ccgcagctgg acgtcctgga gaaggccaag ctgttcgtct cccacggcgg catgaactcc 900

accatggagg ccatgaacgc cggcgtcccg ctggtcgtcg tcccgcagat gcacgagcag 960

gaggtcaccg ccaagcgcgt cgacgagctg ggcctgggcg tccacctgcc gccgaaggag 1020

gtcaccgtcg cccgcctgca gaaggacgtc cagaacgtct acggcgacaa gaacatcctg 1080

tcccgcgtca agtccatgca gcagaagatc gaggagaccg gcggcccgaa gcaggccgtc 1140

tgcgccatcg aggagttcct gaagaccgcc gccgtcgaga actaa 1185

<210> 1

<211> 394

<212> PRT

<213> Bacillus methylotrophicus (B. methylotrophicus B-9987)

<400> 1

Met Arg Lys Thr His Ile Ala Ile Ile Asn Val Ala Ala His Gly His

1 5 10 15

Val Asn Pro Thr Leu Pro Val Ala Glu Glu Leu Val Asn Arg Gly Tyr

20 25 30

Lys Val Thr Phe Ala Thr Thr Glu Glu Phe Glu Ala Ser Val Ala Lys

35 40 45

Thr Gly Ala Ile Pro Val Leu Tyr Arg Thr Ser Ile Lys Ala Asp Pro

50 55 60

Glu Thr Ile Lys Glu Arg Val Asn Lys Asn Asp Ala Phe Val Met Phe

65 70 75 80

Leu Glu Glu Ala Val Glu Val Leu Pro Gln Leu Glu Glu Leu Tyr Lys

85 90 95

Asp Asp Leu Pro Asp Ile Val Leu Phe Asp Phe Leu Ala Leu Ala Gly

100 105 110

Arg Leu Phe Ala Asp Asn Gly Gly Ile Pro Ala Val Lys Phe Cys Pro

115 120 125

Ser Tyr Ala Met Asn Glu Tyr Phe Gln Leu Gly Arg Asp Glu Glu Thr

130 135 140

Leu Glu Ala Ala Lys Gln Ala Ile Glu Glu Tyr Gln Glu Gln Ile Glu

145 150 155 160

Asn Glu Gln Leu Lys Arg Met Thr Met Glu Glu Phe Phe Met Pro Glu

165 170 175

Lys Leu Asn Ile Val Phe Met Pro Arg Ala Phe Gln Pro Lys Gln Glu

180 185 190

Thr Phe Asp Glu Arg Phe Cys Phe Val Gly Pro Ser Leu Gly Glu Arg

195 200 205

Thr Asn Thr Gly Ser Leu Glu Ile Asp Ala Ala Asp Asp Arg Pro Leu

210 215 220

Met Leu Ile Ser Leu Gly Thr Ala Phe Asn Ala Trp Pro Glu Phe Tyr

225 230 235 240

Arg Met Cys Ile Asp Ala Phe Arg Asp Ser Asp Trp Arg Val Ile Met

245 250 255

Ser Val Gly Thr Thr Ile Asp Pro Glu Ser Phe Ser Asp Val Pro Asp

260 265 270

His Phe Ile Ile Arg Gln His Val Pro Gln Leu Asp Val Leu Glu Lys

275 280 285

Ala Lys Leu Phe Val Ser His Gly Gly Met Asn Ser Thr Met Glu Ala

290 295 300

Met Asn Ala Gly Val Pro Leu Val Val Val Pro Gln Met His Glu Gln

305 310 315 320

Glu Val Thr Ala Lys Arg Val Asp Glu Leu Gly Leu Gly Val His Leu

325 330 335

Pro Pro Lys Glu Val Thr Val Ala Arg Leu Gln Lys Asp Val Gln Asn

340 345 350

Val Tyr Gly Asp Lys Asn Ile Leu Ser Arg Val Lys Ser Met Gln Gln

355 360 365

Lys Ile Glu Glu Thr Gly Gly Pro Lys Gln Ala Val Cys Ala Ile Glu

370 375 380

Glu Phe Leu Lys Thr Ala Ala Val Glu Asn

385 390

Claims (4)

1. Use of a glycosyltransferase sbmGT1, the nucleotide sequence of the glycosyltransferase sbmGT1 being as set forth in SEQ ID NO:2, characterized in that: the glycosyltransferase sbmGT1 is applied to the preparation of glycosylation modified piericins compounds.

2. Use according to claim 1, characterized in that: specifically, the glycosyltransferase sbmGT1 transfers and combines glycosyl of glycosyl donor to the piericidin compound; the glycosyl donor is UDP-D-Glu.

3. Use according to claim 1, characterized in that: the piericidin compound is piericidin A1; the glycosylation modification is to carry out glycosylation modification on the 10-position hydroxyl and the 4' -position hydroxyl of the piericidin A1 compound.

4. Use according to claim 1, characterized in that: the method specifically comprises the following steps: (1) cloning a nucleotide sequence encoding the glycosyltransferase sbmGT1 of claim 1 into an integrative vector to construct an integrative expression vector; (2) the expression vector is transferred into streptomycesS. youssoufiensisOUC6819 for high expression; (3) and purifying to obtain the glycosylated piericins compound.

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