Coding gene of carbamoyltransferase and application thereof

Abstract

The invention discloses a coding gene of carbamoyltransferase VtdB and an application of the coding gene. The amino acid sequence of the carbamoyltransferase VtdB is as shown in SEQ ID NO.2, and the nucleotide sequence of the coding gene of the carbamoyltransferase VtdB is as shown in SEQ ID NO.1. According to the invention, targeted blocking is carried out on the carbamoyltransferase gene vtdB, so that the obtained gene blocking mutant strain does not produce the melanocidin A any more, and meanwhile, the melanocidin B and the melanocidin X can be produced. Then, the carbamoyltransferase VtdB is used as a catalyst, and the carbamoyltransferase VtdB can be used for catalyzing the melanocidin B and carbamoyl phosphoric acid in vitro to synthesize the melanocidin A. Functional identification of the vtdB has practical guiding significance on large-scale production of macrolide antibiotics, namely the melanocidin A, the melanocidin B and the melanocidin X, and the vtdB can be applied to the fields of biology, medicine, fine chemical engineering, agriculture and the like.

Description

Technical Field

[0001] This invention belongs to the field of microbial physiology and biochemistry, specifically relating to the encoding gene of carbamoyltransferase vtdB and its application. Background Technology

[0002] Carbamylation is a widespread biosynthetic modification found in microorganisms, plants, and animals, and is a key type of post-biosynthetic modification. In the biosynthesis of secondary metabolites, carbamylated transferases participate in the synthesis and modification of natural products by catalyzing the transfer of carbamylated groups from donor molecules to acceptor molecules, significantly affecting important properties such as antitumor activity, antibacterial activity, cytotoxicity, and yield. Carbamylation typically relies on metal ions such as iron and magnesium to stabilize intermediates, and the transfer of carbamylated groups can be achieved directly or through an ATP-dependent pathway. Carbamylated transferases exhibit high substrate specificity, but their catalytic sites are relatively conserved, generally located at the C3 or C4 hydroxyl groups of hexoses such as glucose, or at the hydroxyl groups of the non-sugar moiety of antibiotics. In rare cases, it occurs at amino, sulfur, carbon, or phosphorus atoms. Carbamylation can enhance or reduce the biological activity of certain natural products. For example, the antitumor activity of the decarbamylated drug neomycin is significantly reduced, and the cytotoxicity and antitumor activity of uncarbamylated anesamcinolone are also significantly reduced. Some antibiotics, after carbamylation, can enhance their inhibitory effect on bacteria and reduce the incidence of drug-resistant bacteria. Furthermore, carbamylation can regulate the stability and solubility of compound molecules to improve pharmacological properties, increase drug half-life, and enhance affinity for targets. Carbamylation modification also participates in the regulation of metabolic networks. In organisms, the carbamylation pathway can regulate the balance of endogenous metabolites, resulting in stronger adaptability to environmental stresses such as adversity and disease resistance. Carbamylation in bacteria and fungi may affect antibiotic synthesis, helping organisms resist attacks from competing microorganisms by regulating the synthesis of toxic metabolites. In addition, the function of carbamylate transferases can also have a certain impact on the microbial community in the ecosystem. Especially in microbial interactions, carbamylated metabolites may participate in ecological phenomena such as competition, symbiosis, and co-pathogenicity among microorganisms as signaling molecules or toxins. In plant pathogens, carbamylated metabolites are often released as toxins to disrupt the host plant's immune system, thereby promoting pathogen infection.

[0003] Venturicidin (VTD), also known as apotoxin, is a typical 20-membered macrolide antibiotic with a macrocyclic skeleton and glycosylated side chains. It was initially isolated from three different actinomycete strains, Streptomycess p. AA32, AA117, and AA590, and subsequently, a series of compounds containing different substituents were isolated. Mechanistically, venturicidin is a specific FO subunit-directed inhibitor that blocks ATP synthesis in bacteria and fungi. Venturicidin exhibits antitrypanosomiasis, antibacterial, and low cytotoxicity. Furthermore, venturicidin A (VTD-A) can inhibit phosphorylation in rat liver mitochondria and inhibit Tetrahymena mitochondrial ATPase, demonstrating antimalarial activity. VTD-A can specifically enhance the resistance of aminoglycoside drugs such as gentamicin to multidrug-resistant bacterial pathogens, assisting in rapid bactericidal action. Although VTD-A exhibits significant toxicity to human embryonic kidney cells in vitro, the compound is highly tolerable when administered in vivo; the highest reported inhibitory concentration is 400 mg / kg intraperitoneally and 20 mg / kg intravenously. Given its unique mechanism of action and broad biological activity, strychnine shows promise for development as an agricultural antibiotic or antifungal drug.

[0004] Streptomyces In sp. NO1W98, the biosynthesis of sclerotin is achieved through the assembly of a macrolide skeleton via a type I PKS pathway. In another sclerotin-producing strain... Streptomyces In sp. NRRLS-4, a similar macrocyclic skeletal synthesis mechanism exists. To date, functional studies of the aminoacyltransferases (VtdB and VenE) involved in the streptomycin biosynthesis pathway in Streptomyces have not been reported domestically or internationally. Therefore, we... Streptomyces The carbamoyltransferase encoding gene in the geraniin A gene cluster of sp. NO1W98 vtdB The in vivo and in vitro functions of this enzyme have been identified, which is expected to lay the foundation for the subsequent modification, development, engineering and application of this type of enzyme. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides the application of carbamoyltransferase VtdB in the field of enzyme engineering, specifically relating to the carbamoyltransferase gene. vtdB Application in the production of black fungicide.

[0006] The first objective of this invention is to provide a carbamoyltransferase VtdB, the amino acid sequence of which is shown in SEQ ID NO.2.

[0007] A second object of the present invention is to provide a gene encoding the carbamoyltransferase VtdB described above, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0008] A third objective of this invention is to provide the application of the above-mentioned carbamoyltransferase VtdB or the above-mentioned encoding gene in the preparation of the macrolide antibiotic sclerotin, wherein the sclerotin is sclerotin A, sclerotin B, and / or sclerotin X, the amino acid sequence of carbamoyltransferase VtdB is shown in SEQ ID NO.2, and the structural formulas of sclerotin A, sclerotin B, and sclerotin X are shown in formulas (I), (II), and (III), respectively:

[0009] The carbamoyltransferase VtdB provided by this invention is derived from actinomycetes. Streptomyces sp. NO1W98 is a protein that is either (a) or (b) as follows:

[0010] (a) A protein composed of the amino acids shown in SEQ ID NO.2;

[0011] (b) A protein derived from (a) by substitution and / or deletion and / or addition of one or more amino acid residues of the amino acid sequence of (a) and having carbamoyltransferase activity.

[0012] The amino acid sequence shown in SEQ ID NO.2 consists of 597 amino acids and has a molecular weight of approximately 65.80 kDa.

[0013] The carbamoyltransferases mentioned in (a) and (b) above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0014] Preferably, the nucleotide sequence of the encoding gene is shown in SEQ ID NO.1.

[0015] The complementary sequence of positions 1-1794 of the sequence shown in SEQ ID NO.1 can be obtained at any time according to the principle of DNA base complementarity. Furthermore, the nucleotide sequence or a portion thereof of positions 1-1794 can be obtained by polymerase chain reaction (PCR), digestion of the corresponding DNA with a suitable restriction endonuclease, DNA in vitro synthesis techniques, or other suitable techniques. This invention provides a method for obtaining a recombinant DNA vector containing at least a portion of the DNA sequence of positions 1-1794 of the sequence shown in SEQ ID NO.1.

[0016] The nucleotide sequence or partial nucleotide sequence provided by this invention can be obtained from other organisms using polymerase chain reaction (PCR) or by using DNA containing positions 1-1794 of the sequence shown in SEQ ID NO.1 of this invention as a probe for Southern hybridization or other methods. vtdB Similar genes.

[0017] The nucleotide sequence or at least a portion thereof provided by this invention can be modified or mutated in vivo or in vitro, including insertion, substitution or deletion, polymerase chain reaction, error-mediated polymerase chain reaction, site-specific mutation, reconnection of different sequences, directed evolution of different parts of the sequence or homologous sequences from other sources, or mutagenesis by ultraviolet light or chemical reagents, etc.

[0018] Clones containing the nucleotide sequences or at least a portion thereof provided by this invention can be expressed in exogenous hosts using suitable expression systems to obtain the corresponding enzymes or other substances with higher bioactivity or yield. These exogenous hosts include Escherichia coli, Streptomyces, Micromonospora, Pseudomonas, Bacillus, yeast, plants, and animals.

[0019] The amino acid sequence provided by this invention can be used to isolate the desired protein and can be used for antibody preparation.

[0020] Peptides containing the amino acid sequence or at least a portion thereof provided by the present invention may retain biological activity or even have new biological activity after the removal or substitution of certain amino acids, or may improve yield or optimize protein kinetic characteristics or other properties to be obtained.

[0021] Genes or gene clusters containing the nucleotide sequences or at least a portion thereof provided by the present invention can be expressed in a heterologous host and their functions in host metabolism can be understood.

[0022] Genes or gene clusters containing the nucleotide sequences or at least a portion thereof provided by the present invention can be used to construct recombinant vectors through genetic recombination to obtain novel biosynthetic pathways, or can be used to obtain other novel biosynthetic pathways or generate new compounds through insertion, substitution, deletion or inactivation.

[0023] The fourth object of this invention is to provide a method for preparing macrolide antibiotics sclerotin B and / or sclerotin X, which is derived from mutant strain Δ vtdB The mutant strain Δ was prepared by fermentation. vtdB Knockout of carbamoyltransferase gene vtdB The carbamoyltransferase gene vtdB The nucleotide sequence is shown in SEQ ID NO.1.

[0024] Preferably, the mutant strain Δ vtdB The preparation method includes the following steps: cosmid 11H10 containing VTDs biosynthetic gene clusters... vtdB Gene deletion to obtain plasmid del vtdB , de plasmid vtdB via E. coli E. coli ET12567 / pUZ8002 conjugation transfer introduced into wild-type actinomycetes Streptomyces From sp. NO1W98 strain, mutant strain Δ was obtained. vtdB .

[0025] Preferably, the mutant strain Δ vtdB The construction method is as follows:

[0026] 1) Transform cosmid 11H10 containing the biosynthetic gene clusters of VTDs into Escherichia coli. E. coli BW25113 / pIJ790 was used to obtain plasmids containing the target plasmid. E. coli BW25113 / pIJ790 / 11H10 strain was prepared into competent cells;

[0027] 2) Using endonucleases Eco RI and H in The plasmid pIJ773 was digested with d III enzyme and a 1.4 kb DNA fragment containing the transfer origin and apopramine resistance gene was recovered as a PCR template. The PCR product was amplified using primers delVtdB-F: TGCGGCCGCACCTTCTCCGGCCTCGGCGACGCCGCCTTCATTCCGGGGATCCGTCGACC and delVtdB-R: CTCCTTCCTGCCCATCTCGAACACCCGCGCCGCGTTCTCTGTAGGCTGGAGCTGCTTC.

[0028] 3) The PCR product was transferred into the competent cells prepared in step 1) to induce recombination. Positive clones were selected, and plasmids were extracted to obtain the recombinant plasmid delta. vtdB ;

[0029] 4) Deploy the recombinant plasmid vtdB Transform to E. coli In ET12567 / pUZ8002, it is constructed as E. coli ET12567 / pUZ8002 / de lvtdB As donor bacteria for conjugation transfer; wild-type actinomycetes Streptomyces After activation, sp. NO1W98 strain spores were isolated and used as recipient bacteria for conjugation transfer;

[0030] 5) Mix the recipient and donor bacteria thoroughly and spread them on M-ISP4 solid medium without any antibiotics. After bacterial growth, transfer the mixture to M-ISP4 solid medium containing 30-40 µg / mL apopramycin and 45-55 µg / mL trimethoprim. Positive clones are then detected. vtdB -Carbamoyltransferase gene knockout double crossover mutant strain Δ vtdB .

[0031] Preferably, the method for preparing the macrolide antibiotics sclerotin B and / or sclerotin X is as follows:

[0032] 1) Preparation of mutant strain Δ vtdB The fermentation products were extracted with ethyl acetate to obtain a fermentation extract.

[0033] 2) The fermentation extract was mixed with silica gel and methanol, evaporated to dryness, and then packed into a column for separation. Nine fractions, A1-A9, were obtained by elution with dichloromethane / methanol at volume ratios of 100:0, 98:2, 96:4, 94:6, 92:8, 90:10, 80:20, 50:50, and 0:100. Fractions A4 and A5 were collected and combined, then eluted with petroleum ether and ethyl acetate at volume ratios of 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, and 0:10 to obtain eluting fractions B1-B11. Fraction B5 was collected and eluted with petroleum ether and ethyl acetate at volume ratios of 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, and 0:10 to obtain eluting fractions C1-C11.

[0034] 3) Components B7, B8, C7, C8, and C9 were further separated by reversed-phase silica gel column chromatography: pure methanol was used for sample transfer. The sample was mixed into C18 packing material with a particle size of 20-45 μm and a pore size of 100-120 nm at a weight ratio of 1:3-1:5. The mobile phase A of the reversed-phase silica gel column chromatography was an acetonitrile solution containing 0.1% acetic acid (v / v), and the mobile phase B was an aqueous solution containing 0.1% acetic acid (v / v). The flow rate was 12 mL / min, the UV detection wavelength was 203 nm, and the elution program was 0-10 minutes, equilibrating the column with 100% B phase. After connecting the sample column, the elution program was set to 0-100 minutes, 100%-0% B phase; 100-120 minutes, 0% B phase. The eluent was collected after 72-75 minutes.

[0035] 4) Evaporate the eluent obtained after 72-75 minutes to dryness and then perform semi-preparative liquid chromatography to obtain cytotoxic sulfadiazine B and cytotoxic sulfadiazine X: Use a 10×250 mm reverse-flow C18 column, mobile phase A is 0.1% acetic acid aqueous solution (v / v) and mobile phase B is acetonitrile, flow rate is 3 mL / min, UV detection wavelength is 203 nm, elution program is 0-60 minutes, and 50% isocratic elution is performed using phase B.

[0036] Preferably, the fermentation product is extracted with ethyl acetate by centrifuging the fermentation product to obtain a supernatant and a precipitate, extracting the supernatant with ethyl acetate, dissolving the precipitate in acetone and sonicating (50 kHz) and filtering, and then extracting the filtrate with ethyl acetate.

[0037] The fifth objective of this invention is to provide a method for the in vitro preparation of the macrolide antibiotic sclerotin A, which uses the aforementioned carbamoyltransferase VtdB to catalyze the synthesis of sclerotin A from sclerotin B and carbamoyl phosphate.

[0038] Preferably, the reaction system further includes ATP, MgCl2, and pH 8.0 Tris-HCl buffer, with the following concentration ratios: carbamoyltransferase VtdB : nitrofurantoin B : carbamoyl phosphate : ATP : MgCl2 : pH 8.0 Tris-HCl buffer at 8-12 μmol / L : 1-3 mmol / L : 1-3 mmol / L : 1-3 mmol / L : 1-3 mmol / L : 48-52 mmol / L. The reaction temperature is 25℃-55℃, and the reaction time is 10-15 h.

[0039] Preferably, 10 μmol / L carbamoyltransferase VtdB, 2 mmol / L methamidophos B, 2 mmol / L carbamoyl phosphate, 2 mmol / L ATP, 2 mmol / L MgCl and 50 mmol / L pH8.0 Tris-HCl buffer are reacted at 28℃-50℃ for 12 h, wherein the methamidophos B is prepared by the above method.

[0040] The gene and protein information of carbamoyltransferase VtdB provided by this invention can be used to produce the tetrahydroβ-carbamoyl skeleton, enhancing our understanding of carbamoyltransferases and providing a material and theoretical basis for further genetic modification. The gene and protein provided by this invention can also be used to find and discover compounds, genes, or proteins that can be used in medicine, industry, or agriculture.

[0041] This invention targets actinomycetes. Streptomyces The carbamoyltransferase gene in the biosynthesis gene cluster of strychnine in sp. NO1W98 vtdBTargeted blocking was performed using PCR-targeting, resulting in gene-blocking mutants that no longer produced cypermethrin A, but were able to produce cypermethrin B and cypermethrin X. Subsequently, the carbamoyltransferase gene was cloned. vtdB The gene was ligated into the expression vector pET28a(+) and transformed into *E. coli* BL21(DE3). After cultivation and induction of expression, the purified carbamoyltransferase VtdB was used as a catalyst to catalyze the synthesis of sclerotin A from carbamoyl phosphate and sclerotin B in vitro. Thus, VtdB was identified both in vivo and in vitro as possessing carbamylation function and is a carbamoyltransferase. The functional identification of the vtdB gene has practical guiding significance for the large-scale production of macrolide antibiotics sclerotin A, sclerotin B, and sclerotin X, and can be applied in fields such as biology, medicine, fine chemicals, and agriculture.

[0042] The present invention Streptomyces sp. NO1W98 strain, plasmid 11H10 and plasmid pIJ773 are disclosed in non-patent literature ( Streptomyces Isolation and identification of the biosynthetic gene cluster of styraxin from sp. NO1W98. Zhang Shaofei, Zhang Yuan, Shen Chuanpu, Chen Qi. Acta Microbiologica Sinica, 2020, 60, 2461–2474. The applicant also holds and will make available to the public for 20 years from the date of application. Attached Figure Description

[0043] Figure 1 It is the chemical structure of macrolide antibiotics melanocytin A (1), melanocytin B (2) and melanocytin X (3).

[0044] Figure 2 It is the chemical reaction formula of venturicidin B and carbamoyl phosphate catalyzed by carbamoyltransferase VtdB to produce venturicidin A.

[0045] Figure 3 yes Streptomyces The strychnine biosynthesis gene cluster in sp. NO1W98 vtdB HPLC analysis of the fermentation extract of the mutant strain obtained after genetic modification in fermentation medium: i: wild type Streptomyces sp. NO1W98; ii–iv: three Δ vtdB Mutant strain; iv: Standard of cypermethrin A; v: Standard of cypermethrin B.

[0046] Figure 4This is the SDS-PAGE analysis result of purified VtdB. Lane 1 indicates the standard protein marker; lane 2 is the purified VtdB protein.

[0047] Figure 5 This is an HPLC chromatogram of the reaction of carbamate B and carbamoyl phosphate as substrates with carbamoyltransferase VtdB to produce carbamate A. i: Standard of carbamate A; ii: Standard of carbamate B; iii–v: Enzymatic reaction of carbamoyltransferase VtdB at 28℃, 37℃ and 50℃ respectively; vi: Control experiment, VtdB inactivated by boiling.

[0048] Figure 6 and Figure 7 It is compound 2. 1 H and 13 The results of the nuclear magnetic resonance spectrum of C.

[0049] Figure 8 and Figure 9 It is compound 3. 1 H and 13 The results of the nuclear magnetic resonance spectrum of C.

[0050] Figure 10 , Figure 11 and Figure 12 The high-resolution mass spectrometry results for compounds 1, 2, and 3 are shown in order. Compound 1: HR-ESI-MS m / z [MH] - = 748.46179 (cacld for C) 41 H 66 NO 11 Compound 2: HR-ESI-MS m / z [MH] (748.4641); Compound 3: HR-ESI-MS m / z [MH] - = 705.45636 (cacld for C) 40 H 65 O 10 Compound 3: HR-ESI-MS m / z [MH] (705.4583); - =575.39514 (cacld for C) 34 H 55 O7, 575.3953).

[0051] Figure 13 and Figure 14 It is compound 1 1 H and 13 The results of the nuclear magnetic resonance spectrum of C. Detailed Implementation

[0052] The following examples are further illustrations of the present invention and are intended to help understand the invention, but are not intended to limit the invention.

[0053] Example 1: vtdB Obtaining a gene-disrupted mutant strain containing the gene (sequence SEQ ID NO.1, 1-1794).

[0054] In vitro knockout mutants were obtained using PCR-targeting. Based on the obtained biosynthetic gene cluster sequence of VTDs (accession number: MN914689.1), the PCR-targeting system reported in the literature was used (Gust B, ChallisGL, Fowler K, Kieser T, Chater KF. PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soilodor geosmin). PNAS . 2003, 100(4): 1541-1546.), Design a pair vtdB The gene knockout primers (delVtdB-F: TGCGGCCGCACCTTCTCCGGCCTCGGCGACGCCGCCTTCA TTCCGGGGATCCGTCGACC; delVtdB-R: CTCCTTCCTGCCCATCTCGAACACCCGCGCCG CGTTCTCTGTAGGCTGGAGCTGCTTC). Then, according to the PCR-targeting method, an in vitro knockout plasmid was constructed and then transformed into the conjugated donor bacteria. The specific steps are as follows: (1) Plasmid 11H10 containing the biosynthetic gene cluster of VTDs (published in the reference: Streptomyces Isolation and identification of the biosynthetic gene cluster of nitrofurantoin from sp. NO1W98. Zhang Shaofei, Zhang Yuan, Shen Chuanpu, Chen Qi. Acta Microbiologica Sinica, 2020, 60, 2461–2474) transformed into Escherichia coli. E. coli BW25113 / pIJ790 was used to obtain plasmids containing the target plasmid. E. coli BW25113 / pIJ790 / 11H10 strain was induced to express λ-red recombinant system using LB solid medium containing 10 mmol / L L-arabinose, and then prepared as competent cells for use. (2) Endonuclease Eco RI and Hind III digestion of plasmid pIJ773 (published in reference: StreptomycesIsolation and Identification of Biosynthetic Gene Cluster of Acetaminophen from sp. NO1W98. Zhang Shaofei, Zhang Yuan, Shen Chuanpu, Chen Qi. Acta Microbiologica Sinica, 2020, 60, 2461–2474). A DNA fragment containing the transfer origin and apopramine resistance gene of approximately 1.4 kb was recovered and used as a PCR template. The 1.4 kb PCR product was amplified by PCR using delVtdB-F and delVtdB-R primers. The 50 µL PCR reaction system consisted of: 3 U high-fidelity DNA polymerase, 5 µL 10×Buffer, 0.5 mmol / L dNTPs, 2.5 µL DMSO, 0.5 µmol / L primers, approximately 1 ng DNA template, and water added to a final volume of 50 µL. The PCR reaction conditions were as follows: pre-denaturation at 94℃ for 5 min; amplification cycles were 94℃ denaturation for 45 s, 58℃ annealing for 45 s, 72℃ extension for 90 s, 30 cycles; and a final extension at 72℃ for 10 min. The 1.4 kb PCR product was recovered and purified for later use. (3) The PCR product from step (2) was transferred into the competent cells prepared in step (1) to induce recombination. The cells were then plated on LB selection plates (containing 100 µg / mL ampicillin, 50 µg / mL kanamycin, and 50 µg / mL apopramine) and cultured overnight at 37℃. Positive single clones were picked from the plates, and plasmids were extracted. The recombinant plasmid was named del. vtdB The plasmid in ​ Part of the gene was replaced by the transfer origin and the apopramine resistance gene. (4) The constructed recombinant mutant plasmid de ​ Transform to ​ In ET12567 / pUZ8002, it is constructed as ​ ET12567 / pUZ8002 / del ​ , as donor bacteria for conjugation transfer.

[0055] Wild-type actinomycetes ​ strain NO1W98 was streaked in M-ISP4 solid medium (10 g soluble starch, 0.5 g yeast extract, 1 g peptone, 1 g NaCl, 1 g MgSO4·7H2O, 2 g (NH4)2SO4, 1 g K2HPO4, 2 g CaCO3, 20 g agar powder, water added to 1 L, pH adjusted to 7.2, and sterilized) for 3-5 days. The resulting spores were collected using sterile cotton swabs and placed in TSB medium. The spores were dispersed by vortexing. Mycelium and spores were separated by filtration with sterile cotton swabs. The spores were suspended in 5 mL of TSB medium, heat-shocked at 50°C for 10 min, and then germinated at 28°C for 4 h, serving as the recipient bacteria for conjugation transfer. Donor bacteria. ​ET12567 / pUZ8002 / del ​ The cells were grown at 37°C to OD in 50 mL of LB liquid medium containing 50 µg / mL kanamycin, 25 µg / mL chloramphenicol, and 50 µg / mL apopramine. 600 When the value was approximately 0.8, the bacterial cells were collected by centrifugation (4000 r / min, 10 min), washed three times with LB broth, and suspended in 300 µL of LB liquid medium as donor bacteria for conjugation transfer. 400 µL of the recipient bacteria and 100 µL of the donor bacteria were mixed thoroughly and spread onto M-ISP4 solid medium without any antibiotics. After drying, the mixture was incubated at 28°C for 20 h. The plates were then removed, covered with water containing antibiotics (final concentrations of 35 µg / mL apopramycin and 50 µg / mL trimethoprim), dried, and placed in a 28°C incubator for 3 days before observation.

[0056] After small bacteria grow on the conjugation transfer plate, they are transferred with sterile toothpicks to M-ISP4 plates containing 35 µg / mL apopramycin and 50 µg / mL trimethoprim. After incubation at 28°C for 2–3 days, genomic DNA is extracted from each mutant strain. Positive clones are obtained by PCR detection using detection primers (tsVtdB-F: TGACCAGGTGGTCGATGTCG; tsVtdB-R: GGACGAGAAGCTGATCACCG). ​ -Carbamoyltransferase gene knockout double crossover mutant strain (Δ ​ ).

[0057] Example 2: ​ wild sp. NO1W98 and mutant strain (Δ) ​ Fermentation and HPLC detection of )

[0058] Actinomycetes ​ sp. NO1W98 wild-type fungus and three mutant strains (Δ ​ Streak the inoculum onto M-ISP4 solid medium and incubate at 28°C for 7 days. Scrape approximately 2 cm of the culture medium. 2The culture was inoculated into 50 mL of B1 liquid fermentation medium (10 g soybean flour, 10 g glucose, 15 g soluble starch, 5 g yeast powder, 5 g NaCl, 3 g CaCO3, 1 L H2O, pH 7.0) in a 250 mL Erlenmeyer flask and cultured at 28 °C for 7 days. Then, an equal volume of ethyl acetate was added, and the cells were sonicated (50 kHz) for 5 min to disrupt the cells. The mixture was then allowed to separate into layers. The ethyl acetate extract was separated from the aqueous phase, and the ethyl acetate was evaporated to dryness using a rotary evaporator. The crude extract was dissolved in 1 mL of methanol to form a sample, which was then analyzed by HPLC. The detection conditions were: Agilent Zorbax SB-C18 (150 × 4.6 mm, 5 μm) reversed-phase column; mobile phase A: ddH2O containing 0.1% acetic acid (v / v); mobile phase B: 100% acetonitrile containing 0.1% acetic acid (v / v); flow rate: 0.75 mL / min; detection wavelengths: 203 nm and 273 nm. HPLC program: 0–20 min, 40%–100% B phase; 20–25 min, 100% B phase; 25–26 min, 100%–40% B phase; 26–30 min, 40% B phase. Weigh 1 mg each of the standards of cypermethrin A and cypermethrin B, dissolve them separately in 1 mL of DMSO, dilute 100 times with methanol, and take 10 μL for HPLC analysis, using the same method as before.

[0059] Specific results are as follows ​ As shown, knocked out ​ -Carbamoyltransferase gene, obtained mutant strain Δ ​ Fermentation could not produce cypermethrin A, but it did produce two new compounds, 2 and 3. Subsequent large-scale exploration, isolation, purification, and structural identification led to their identification as cypermethrin B and cypermethrin X, respectively. ​ For details, please refer to Example 3.

[0060] Example 3: Mutant strain Δ ​ Large-scale fermentation and isolation and structural identification of compounds 2 and 3

[0061] mutant strain Δ ​ Large-scale fermentation is the same as in Example 2 (mutant strain Δ) ​ After activation, scrape off about 2 cm. 2 The culture was inoculated into 50 mL of B1 liquid fermentation medium in a 250 mL Erlenmeyer flask and cultured at 28 °C for 7 days. A total of 10 L of fermentation product was obtained. The mixture was centrifuged at 4000 rpm for 30 minutes to separate the supernatant and precipitate. An equal volume of ethyl acetate was added to a separatory funnel to extract the secondary metabolites from the supernatant. Extraction was repeated 3-4 times until essentially colorless. The upper organic phase was collected and evaporated to dryness.

[0062] Add an equal volume of acetone to the precipitate obtained by centrifugation, sonicate (50 kHz) for 15 minutes, filter through gauze to collect the liquid, and repeat this operation 3 times until it is basically colorless or slightly yellow. Rotate the collected filtrate to collect the secondary metabolites. Add an equal volume of ethyl acetate for back-extraction, shake to mix, and allow to stand for about 6 hours. Use a rotary evaporator to recover the ethyl acetate and collect the fermentation extract. Repeat this operation at least 7 times until the extract is nearly colorless.

[0063] The total amount obtained after extraction with ethyl acetate and acetone was obtained separately. ​ sp.NO1W98 / ΔvtdB fermentation extract 6.7 g.

[0064] 15 g of silica gel and 50 mL of methanol were added to the above fermentation extract and mixed. After evaporation to dryness, the mixture was packed into a column for separation. For the first time, dichloromethane and methanol were used as eluents. Elution was performed at volume ratios of C:M (dichloromethane:methanol) = 100:0, C:M = 98:2, C:M = 96:4, C:M = 94:6, C:M = 92:8, C:M = 90:10, C:M = 80:20, C:M = 50:50, and C:M = 0:100 to obtain the corresponding nine fractions A1, A2, A3, A4, A5, A6, A7, A8, and A9. After merging components A4 and A5, which contain the highest concentrations of the target compound, petroleum ether and ethyl acetate were used as eluents. Elution was performed at volume ratios P:E (petroleum ether:ethyl acetate) of 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, and 0:10 to obtain the corresponding 11 components B1, B2, B3, B4, B5, B6, B7, B8, B9, B10, B... 11; Similarly, petroleum ether and ethyl acetate were used as eluents to separate and purify fraction B5, which contained a relatively high amount of the target compound. Elution was performed at volume ratios of P:E = 10:0, P:E = 9:1, P:E = 8:2, P:E = 7:3, P:E = 6:4, P:E = 5:5, P:E = 4:6, P:E = 3:7, P:E = 2:8, P:E = 1:9, and P:E = 0:10, yielding 11 corresponding fractions: C1, C2, C3, C4, C5, C6, C7, C8, C9, and C11. Fractions B7, B8, C7, C8, and C9 contained a significant amount of the target compound, but each fraction still exhibited numerous impurity peaks. Further separation was then performed using reversed-phase silica gel column chromatography.

[0065] 5 mL of pure methanol was used to transfer the sample. The sample was mixed into C18 packing material with a particle size of 20-45 μm and a pore size of 100 Å at a weight ratio of 1:4, and heated in a 40°C water bath to completely evaporate the methanol. The mobile phase for reversed-phase silica gel column chromatography was an acetonitrile solution containing 0.1% acetic acid (phase A) and an aqueous solution containing 0.1% acetic acid (phase B), with a flow rate of 12 mL / min, a UV detection wavelength of 203 nm, and an elution program of 0-10 minutes, equilibrating the column with 100% phase B. After connecting the sample column, the elution program was set to 0-100 minutes, 100%-0% phase B; and 100-120 minutes, 0% phase B. 200 µL of the eluent was collected from 0-72 minutes, 72-75 minutes, and 75-110 minutes, respectively, and directly used for HPLC analysis, following the same detection procedure as above (Example 2). B7, B8, and C7, C8, and C9 were subjected to reverse-phase silica gel column chromatography separately. A relatively pure target compound was collected in an acetonitrile eluent of approximately 75% concentration at 72-75 minutes. After rotary evaporation, a total of 335.44 mg of the target compound was obtained from the five fractions (B7, B8, C7, C8, and C9). However, the purity of the compound was still insufficient for identification by high-resolution mass spectrometry and nuclear magnetic resonance. Therefore, the compound was further separated and prepared by semi-preparative liquid chromatography.

[0066] Preparative HPLC method and high-performance liquid chromatography (HPLC) analysis. Semi-preparative method: A 10×250 mm reverse-flow C18 column was used. The mobile phase was 0.1% acetic acid aqueous solution (phase A) and acetonitrile (phase B), with a flow rate of 3 mL / min. The UV detection wavelength was 203 nm, and the elution program was 0-60 minutes, 50% phase B, column temperature 30°C, and isocratic elution. Semi-preparative HPLC prepared components B7, B8, C7, C8, and C9. Peaks corresponding to target compound 2 were detected at 22 min for all five components, and peaks corresponding to target compound 3 were detected at 48.2 min. 200 µL of each eluent was collected and directly analyzed by HPLC (detection program same as in Example 2). After confirming purity, the eluent was evaporated to dryness, yielding pure compound 2 (96 mg) and compound 3 (35 mg), respectively. NMR and HRESIMS analyses were performed. The obtained H and C spectral data are shown in Tables 1 and 2, and the corresponding NMR chromatograms are shown in Tables 2 and 3, respectively. ​ -9, HRESIMS spectrum see ​ and ​ Therefore, compounds 2 and 3 were identified as cypermethrin B and cypermethrin X, respectively, with the following structures: ​ As shown.

[0067] Table 1. 1 H and 13C NMR spectroscopic data for compounds 2 in DMSO- d 6. ; .

[0068] Table 2. 1 H and 13 C NMR spectroscopic data for compounds 3 in DMSO- d 6. ; .

[0069] Example 4: Preparation of carbamoyltransferase VtdB

[0070] Cloning, expression, affinity chromatography purification, SDS-PAGE electrophoresis analysis, and Bradford assay of carbamoyltransferase VtdB:

[0071] Target gene ​ amplification with ​ Using sp. NO1W98 as a template, design primer pair vtdB-F: CGCCATATG ​ ;vtdB-R:CCCAAGCTT ​ Selecting enzyme cleavage sites ​ I and ​ III. The vector pET-28a(+) was double-digested, and the target gene was ligated to the vector using T4 ligase and transformed into DH5α competent cells. After overnight culture at 37°C, single clones were picked for sequencing verification, yielding correctly sequenced pET28a(+) / ​ Plasmids were transformed to obtain recombinant strains. ​ BL21 / pET28a(+) / ​ .

[0072] Recombinant strains ​ BL21 / pET28a(+) / ​ After overnight culture in LB liquid medium, inoculate 1% (v / v) into 300 mL of LB culture medium in 1 L Erlenmeyer flasks (15 flasks in total), and incubate at 37°C on a shaker at 200 r / min until OD. 600When the pH reached approximately 0.6, isopropyl-β-d-thiogalactopyranoside (IPTG) was added to the culture to a final concentration of 0.04 mmol / L. The culture was then incubated at 16°C and 160 rpm for 20 h. The cells were collected by centrifugation at 4000 rpm for 10 min, washed twice with 20 mL of buffer (20 mmol / L Tris-HCl, pH 8.0, 200 mM NaCl), and finally resuspended in 15 mL of Binding Buffer (20 mmol / L Tris-HCl, pH 8.0, 200 mmol / L NaCl, 5 mmol / L Limidazole, pH 8.0). The cells were lysed by sonication at 0°C (20 kHz, 5 s on, 5 s off, 60 cycles, total 10 min). After lysis, the cells were centrifuged at 4°C and 10000 rpm for 40 min. Then, the protein was purified according to the following procedure: (1) Column packing: The supernatant was added to the Ni-NTA affinity column, and the filtrate was collected; (2) Washing: 10 mL Binding Buffer (approximately 10 column volumes) was added, and the filtrate was added to 15 mL Washing Buffer (approximately 18 column volumes, 20 mmol / L Tris-HCl, pH 8.0, 200 mmol / L NaCl, 20 mmol / L imidazole) to wash away the contaminating proteins bound to the Ni-NTA column; (3) Elution: 3.5 mL of Elution Buffer 1 (20 mmol / L Tris-HCl, pH 8.0, 500 mmol / L NaCl, 250 mmol / L imidazole) was added to elute the target protein from the Ni-NTA column. To prevent the target protein from over-binding to the Ni-NTA column, 1 mL of Elution Buffer 2 (20 mmol / L Tris-HCl, pH 8.0, 200 mmol / L NaCl, 500 mmol / L imidazole) was added to the column. (4) Concentration: Transfer the 3.5 mL filtrate eluted with Elution Buffer I to a 15 mL 10 kD ultrafiltration tube, centrifuge at 2500 r / min and 4℃ for 20 min, and concentrate to less than 2.5 mL; (5) Desalting: Transfer the concentrated protein to a PD-10 desalting column for desalting, wash with 2.5 mL Storage Buffer (volume ratio 30% glycerol, 20 mmol / L Tris-HCl, pH 8.0) to wash off the target protein, and then concentrate it with a 10 kD ultrafiltration tube. Store the concentrated target protein at -80℃ for later use, thereby obtaining the gene. ​The encoded protein is VtdB (its amino acid sequence is shown in SEQ ID NO.2).

[0073] Take 5 μL of the purified protein VtdB, add water to make up to 20 μL, add 5 μL of 5×SDS PAGE Loading Buffer, mix thoroughly, boil in water for 10 min, take 5 μL of the sample for SDS PAGE electrophoresis (Tris-Glycin buffer system), and simultaneously, dilute the purified protein by 1, 2, and 10 times, take 5 μL of each, add water to make up to 20 μL, add 1 mL of 1×Bio-Rad Protein Assay reagent, react for 10 min, measure the absorbance at 595 nm, and calculate the concentration of carbamoyltransferase VtdB by comparing with the Bradford standard protein concentration curve.

[0074] SDS-PAGE of carbamoyltransferase VtdB protein as shown in Figure 1 ​ As shown, the concentration of the purified VtdB protein was approximately 35 μM.

[0075] Example 5: In vitro reaction of carbamoyltransferase VtdB

[0076] Next, the in vitro reaction of carbamoyltransferase VtdB was tested under the following conditions:

[0077] 2 mmol / L styraxin B + 2 mmol / L carbamoyl phosphate (CP) + 2 mmol / L ATP + 2 mmol / L MgCl2 + 10 μmol / L carbamoyltransferase VtdB + 50 mmol / L pH 8.0 Tris-HCl buffer were reacted at 28℃, 37℃, and 50℃ for 12 h each, with the reaction of boil-inactivated carbamoyltransferase VtdB at 37℃ serving as a control. The reaction was terminated with 500 μL ethyl acetate. After thorough mixing, the mixture was centrifuged at 10,000 rpm for 5 min, and the supernatant was collected. This process was repeated three times. All supernatants were evaporated to dryness and dissolved in 200 µL methanol. After centrifugation at 12,000 rpm for 20 min, 30 μL of the supernatant was collected for HPLC analysis. The product was analyzed using a Waters Alliance e2695 HPLC system, and the product was preliminarily identified by high-resolution mass spectrometry. HPLC detection conditions were as follows: an Agilent Zorbax SB-C18 (150 × 4.6 mm, 5 μm) reversed-phase column; mobile phase A was 0.1% (v / v) aqueous acetic acid; mobile phase B was 99.9% (v / v) acetonitrile + 0.1% acetic acid; flow rate was 0.75 mL / min; detection wavelengths were 203 nm and 273 nm. HPLC program: 0–20 min, 40%–100% B phase; 20–25 min, 100% B phase; 25–26 min, 100%–40% B phase; 26–30 min, 40% B phase.

[0078] The HPLC analysis results of the conversion of methamidophos B and carbamoyl phosphate to methamidophos A under the catalysis of carbamoyltransferase VtdB are as follows: ​ As shown, ​ In the diagram, i: standard of cypermethrin A; ii: standard of cypermethrin B; iii–v: enzyme reaction of carbamoyltransferase VtdB at 28℃, 37℃, and 50℃ respectively; vi: control experiment, VtdB inactivated by boiling. Where 1 represents... ​ Compound 1 in the formula can be obtained by HPLC preparation followed by high-resolution mass spectrometry identification; the high-resolution mass spectra of compound 1 are shown below. ​ As shown, compound 1 1 H and 13 The results of the nuclear magnetic resonance spectrum of C are as follows: ​ and ​ As shown in Table 3, the NMR data of compound 1 are presented. The structure of compound 1 was thus identified as follows: ​ As shown in Equation 1.

[0079] Table 3. 1 H and13 C NMR spectroscopic data for compounds 1 in DMSO- d 6.

[0080] Depend on ​ It can be seen that carbamoyltransferase VtdB is responsible for the reaction between sclerotin B and carbamoyl phosphate, thereby producing compound 1 ( ​ Compound 1 belongs to the macrocyclic lactone class of compounds.

[0081] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

[0082] SEQ ID NO.1

[0083] ​​

[0084] SEQ ID NO.2

[0085] MIILGYNGFSQIAELFGRLYGYTADSVDRHSFLGHDAAAALFVDGELVAAVEEERMNRQKKTTAFPANAMRWCLEGAGISYEDVDYYAFGWNFTAEFADAAITGLASAPIPPEYKFQAIGSFGELWNGALGRTALIEDFTRHTGYALPDEKLITVPHHRAHLACGRTFSGLGDAAFLINDGQAEADSAIMGEVRDGKVEVFERFTIDAKNSLAQLFANITRYLGFTPNNDEYKVMGLAGFGKAPDEQDNPLLTKVVTLEEGGRYSLALANDPRGPRAYDPLFDELFDGGNDDNRQEFDF RVRVACAAQQVIEAVTAHQLRALAEATELRDLIFEGGLALNCVNNTKLLEELPFTRVEVSFGASDPGVSIGAAAHVAREKSVALTPTESPYLGPEFGEDIRATLEEYTSSVTWEQLPSDEVVGKTAELLTGKTVIGWFQGRTEYGPRALGNRSILANPSYADMKDVINNRVKHREPFRPFAPIVLEENAARVFEMGRKERSPYMTFVFPVRPEYTEKIAAATHVDATSRIQTVTEDSNPRLAALLREFTSRTDVPCLVNTSFNVAGEPIVCSPKDAVECFLGTDIDHLVIGDFLVSKR

Claims

1. Carbamoyltransferase VtdB, characterized in that, The amino acid sequence is shown in SEQ ID NO.

2.

2. The gene encoding the carbamoyltransferase VtdB as described in claim 1, characterized in that, The nucleotide sequence is shown in SEQ ID NO.

1.

3. The application of the carbamoyltransferase VtdB as described in claim 1 or the encoding gene as described in claim 2 in the preparation of the macrolide antibiotic sclerotin, characterized in that, The cypermethrin is cypermethrin A, cypermethrin B, and / or cypermethrin X, and the structural formulas of cypermethrin A, cypermethrin B, and cypermethrin X are shown in formula (I), formula (II), and formula (III), respectively: 。 4. A method for preparing macrolide antibiotics sclerotin B and / or sclerotin X, characterized in that, It is from the mutant strain Δ vtdB The mutant strain Δ was prepared by fermentation. vtdB Knockout of carbamoyltransferase gene vtdB The carbamoyltransferase gene vtdB The nucleotide sequence is shown in SEQ ID NO.

1.

5. The method according to claim 4, characterized in that, The mutant strain Δ vtdB The preparation method includes the following steps: cosmid 11H10 containing VTDs biosynthetic gene clusters... vtdB Gene deletion to obtain plasmid del vtdB , de plasmid vtdB via E. coli E. coli ET12567 / pUZ8002 conjugation transfer introduced into wild-type actinomycetes Streptomyces From sp. NO1W98 strain, mutant strain Δ was obtained. vtdB .

6. The method according to claim 4 or 5, characterized in that, The mutant strain Δ vtdB The construction method is as follows: 1) Transform cosmid 11H10 containing the biosynthetic gene clusters of VTDs into Escherichia coli. E. coli BW25113 / pIJ790 was used to obtain plasmids containing the target plasmid. E. coli BW25113 / pIJ790 / 11H10 strain was prepared into competent cells; 2) Using endonucleases Eco RI and H in The plasmid pIJ773 was digested with d III enzyme and a 1.4 kb DNA fragment containing the transfer origin and apopramine resistance gene was recovered as a PCR template. The PCR product was amplified using primers delVtdB-F: TGCGGCCGCACCTTCTCCGGCCTCGGCGACGCCGCCTTCATTCCGGGGATCCGTCGACC and delVtdB-R: CTCCTTCCTGCCCATCTCGAACACCCGCGCCGCGTTCTCTGTAGGCTGGAGCTGCTTC. 3) The PCR product was transferred into the competent cells prepared in step 1) to induce recombination. Positive clones were selected, and plasmids were extracted to obtain the recombinant plasmid delta. vtdB ; 4) Deploy the recombinant plasmid vtdB Transform to E. coli In ET12567 / pUZ8002, it is constructed as E. coli ET12567 / pUZ8002 / de lvtd B, as a donor bacterium for conjugation transfer; wild-type actinomycetes Streptomyces After activation, sp. NO1W98 strain spores were isolated and used as recipient bacteria for conjugation transfer; 5) Mix the recipient and donor bacteria thoroughly and spread them on M-ISP4 solid medium without any antibiotics. After bacterial growth, transfer the mixture to M-ISP4 solid medium containing 35 µg / mL apopramycin and 50 µg / mL trimethoprim. Positive clones are then detected. vtdB -Carbamoyltransferase gene knockout double crossover mutant strain Δ vtdB .

7. The method according to claim 4, characterized in that, The method for preparing the macrolide antibiotics meliocin B and / or meliocin X is as follows: 1) Preparation of mutant strain Δ vtdB The fermentation products were extracted with ethyl acetate to obtain a fermentation extract. 2) The fermentation extract was mixed with silica gel and methanol, evaporated to dryness, and then packed into a column for separation. Nine fractions, A1-A9, were obtained by elution with dichloromethane / methanol at volume ratios of 100:0, 98:2, 96:4, 94:6, 92:8, 90:10, 80:20, 50:50, and 0:

100. Fractions A4 and A5 were collected and combined, then eluted with petroleum ether and ethyl acetate at volume ratios of 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, and 0:10 to obtain eluting fractions B1-B11. Fraction B5 was collected and eluted with petroleum ether and ethyl acetate at volume ratios of 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, and 0:10 to obtain eluting fractions C1-C11. 3) Components B7, B8, C7, C8, and C9 were further separated by reversed-phase silica gel column chromatography: pure methanol was used for sample transfer. The sample was mixed into C18 packing material with a particle size of 20-45 μm and a pore size of 100-120 nm at a weight ratio of 1:3-1:

5. The mobile phase A of the reversed-phase silica gel column chromatography was an acetonitrile solution containing 0.1% acetic acid (v / v), and the mobile phase B was an aqueous solution containing 0.1% acetic acid (v / v). The flow rate was 12 mL / min, the UV detection wavelength was 203 nm, and the elution program was 0-10 minutes, equilibrating the column with 100% B phase. After connecting the sample column, the elution program was set to 0-100 minutes, 100%-0% B phase; 100-120 minutes, 0% B phase. The eluent was collected after 72-75 minutes. 4) The eluent obtained after 72-75 minutes was evaporated to dryness and then separated by semi-preparative liquid chromatography to obtain cytotoxic sulfadiazine B and cytotoxic sulfadiazine X: A 10×250 mm reverse-directed C18 column was used, the mobile phase A was 0.1% acetic acid aqueous solution (v / v) and the mobile phase B was acetonitrile, the flow rate was 3 mL / min, the UV detection wavelength was 203 nm, the elution program was 0-60 minutes, the column temperature was 30 degrees Celsius, and 50% of the B phase was used for isocratic elution.

8. A method for in vitro preparation of the macrolide antibiotic sclerotin A, characterized in that, The method involves using the carbamoyltransferase VtdB described in claim 1 to catalyze the synthesis of sclerotin B and carbamoyl phosphate into sclerotin A.

9. The method according to claim 8, characterized in that, The reaction system also includes ATP, MgCl2, and pH 8.0 Tris-HCl buffer. The concentration ratio of each component is as follows: carbamoyltransferase VtdB : nitrofurantoin B : carbamoyl phosphate : ATP : MgCl2 : pH 8.0 Tris-HCl buffer is 8-12 μmol / L : 1-3 mmol / L : 1-3 mmol / L : 1-3 mmol / L : 1-3 mmol / L : 48-52 mmol / L. The reaction temperature is 25℃-55℃, and the reaction time is 10-15 h.

10. The method according to claim 8 or 9, characterized in that, The reaction is carried out by reacting 10 μmol / L carbamoyltransferase VtdB, 2 mmol / L methamidophos B, 2 mmol / L carbamoyl phosphate, 2 mmol / L ATP, 2 mmol / L MgCl and 50 mmol / L pH8.0 Tris-HCl buffer at 28℃-50℃ for 12 h, wherein the methamidophos B is prepared by the method described in any one of claims 3-7.

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