Uridine-5apos, uridine-5apos; diphosphate polydeoxygenated diamino sugar as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to uridine-5 '-diphosphate polydeoxy diamino sugar as well as a preparation method and application thereof. According to the invention, uridine-5 '-diphosphate-N-acetyl-glucosamine is used as a substrate to prepare UDP-kasuramine and a new structural analogue UDP-3 ''-hydroxyl-kasuramine of the UDP-kasuramine. The compound can be used as a medical intermediate for preparing commercial agricultural and medical antibiotic kasugamycin and derivatives thereof without / with low toxic and side effects. The method is simple and convenient to operate, mild in condition, environment-friendly and relatively high in conversion rate, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular to uridine-5′-bisphosphate polydeoxydiaminosaccharide, its preparation method and application. Background Technology

[0002] Kasugamycin is an aminoglycoside antibiotic produced by *Streptomyces kasugaensis* and *Streptomyces microaureus*. Its structure consists of three parts: a core skeleton of polydeoxydiaminosaccharide kasugamycin, D-chiral inositol, and an iminoacetic acid side chain. The core skeleton kasugamycin is a 2,3,4,6-tetradeoxy-2,4-diaminopyranose unit.

[0003] Kasugamycin possesses excellent antifungal and antibacterial activity and is currently a commercially available biological pesticide in the agricultural sector. In the pharmaceutical field, early clinical studies have shown that topical kasugamycin is significantly effective in treating burns, dermatological surgeries, and exfoliative dermatitis; systemic application also shows good efficacy in treating urinary tract infections, respiratory infections, and sepsis caused by Pseudomonas aeruginosa and other Gram-negative bacilli. However, injectable kasugamycin hydrochloride was discontinued clinically in 2003 due to its high toxicity. Therefore, understanding the biosynthetic mechanism of kasugamycin and the green bio-manufacturing of its key intermediates is of great significance for subsequent rational modification to reduce its toxicity and enhance its drug-like properties.

[0004] Therefore, using synthetic biology techniques to express recombinant proteins of the gene responsible for the biosynthesis of kasugamycin's core backbone, kasugamine, and then performing a one-pot, multi-enzyme, green, and efficient synthesis of the intermediate kasugamine and its analogues in vitro will greatly enhance the potential of kasugamycin and its derivatives with no or low toxicity, and hopefully propel them back into clinical research. Summary of the Invention

[0005] In view of this, the present invention uses uridine-5′-bisphosphate-N-acetyl-glucosamine (hereinafter referred to as UDP-GlcNAc) as a substrate to obtain a novel uridine-5′-bisphosphate polydeoxydiaminosaccharide compound, uridine-5′-bisphosphate-kasugamine (hereinafter referred to as UDP-kasugamine), and its novel structural analogue, UDP-3”-hydroxy-kasugamine. This new compound can be used as a pharmaceutical intermediate to prepare commercially available agricultural and pharmaceutical antibiotic kasugamycin and its derivatives with no or low toxicity. The method is simple to operate, operates under mild conditions, is environmentally friendly, and has a high conversion rate, showing promising application prospects.

[0006] This invention provides uridine-5′-bisphosphate polydeoxydiaminosaccharide compounds, said compounds comprising the structure as described in Formula 1 and pharmaceutically / pesticide-acceptable salts, esters and prodrugs;

[0007]

[0008] The R includes H and OH;

[0009] When R is H, it is named UDP-Spring Thunderamine, and the structure is shown in Formula 2:

[0010]

[0011] When R is OH, it is named UDP-3”-hydroxy-kasugamine, and the structure is shown in Formula 3:

[0012]

[0013] The present invention also provides a method for preparing the above-mentioned compound. When preparing UDP-kasugamine, the preparation method includes the following steps: using uridine-5′-bisphosphate-N-acetyl-glucosamine as a substrate, a one-pot multi-enzyme reaction is carried out in a buffer system by isomerase KsgQ, deacetylase KsgB, dehydratase KsgD, dehydrogenase KsgR and transaminase KsgC, and the product is obtained after purification;

[0014] When preparing UDP-3”-hydroxy-kasugamine, the preparation method includes the following steps: using uridine-5′-bisphosphate-N-acetyl-glucosamine as a substrate, a one-pot multi-enzyme reaction is carried out in a buffer system by isomerase KsgQ, deacetylase KsgB, dehydratase KsgD and transaminase KsgC, and the product is obtained after purification.

[0015] In this invention, the amino acid sequence of the isomerase KsgQ is shown in SEQ ID NO.1:

[0016] MALRVGIVYGTRPEAIKLAPLVLALDADPGFEPVIITTGQHRDMLDEINELFGLRPRHNLDIMRPGQRLSAMASRIVGELGDPLLDELVDVAVVQGDTSTAFAAAYAAACERIPVAHLEAGLRTGDRFEPFPEEINRRLITQLADLHFAPTADAAGNLLAEGVRSDDVYVTGNTVIDAMHLVLDRPGDSANR ELDAFTEGRQTVLLTMHRRESWGIPMGRVAAAVAELCRSRPTLRFVIPLHPNPEVRRVFRSHLSSLTQVLLCEPLRYSEFIRLMHRAVLVLTDSGGVQEEAPTLGKPVLVLRDRTERPEGIAAGCARLVGTDPALIVKEVGRLLDDPEAYEAMRRPGIVCYGEGDAAARCLEALRERWLSSPDASAGLRTYR;

[0017] The amino acid sequence of the deacetylase KsgB is shown in SEQ ID NO.2:

[0018] MTGHGVKHPSAPETEERDMSSHQALADQTSQVPPEREDRLARALAAWLRGGGTQRADGAYCGWQAPDGRLSYPYPEITGYILTFFAHETLDASERQRADAAASWLADRIDSGDYSCRPDHGDGGVFLFDIGMITHGLLSYGRRTGDQRLLSSGRAAARFLLDHLPTRGHPHPLATGHRPEGTEPTWSNSGSA HLLKLVQALISADGAGVTGAAQAAERLVDTVLADCPPPSAAPVTTCPDSDLISLHAACYAAEGLWIWDTARRRPQARERAVRITEWVWQQQLTGGGFTTYAHRTGGPASDRMQSDVLAQAIRLAKLLDLLRPDGLGSAVSTLANSLHTYDDKAAVLYRPQAAEPHRNSWSSMFAGQALRLCGAEAGLAWHELV;

[0019] The amino acid sequence of the dehydratase KsgD is shown in SEQ ID NO.3:

[0020] MSPTTTHWAGRQVLVTGADGFIGSHLTETLVSRGARVTAVVRRVSAAQVTHRLRNLSAATVDALERVVHVDLAGPSAVDVLGRLEADTWFHLAADAYVPASLDQPADVVRTNVMSTLHVLLAAQQRQPAHLLVTSSSEVYGSQPDAITERHPLEPATPYAASKVA CDRLAWSWHHTYGLPLTIVRPFNSYGPRHVYDAVPLFLARALRGEPITINGSGEQTRDLTFVADTVAGFLALAELPATGETYNIGTGTDHRIIDVARAIVALTGSQSEIVHGPPRSGEVLKLQADPAKLTEATGWRAEYDLARGLADNLVWMREHVETVWPTRS;

[0021] The amino acid sequence of the dehydrogenase KsgR is shown in SEQ ID NO.4:

[0022] MEIIHAGGDGTLLSFGRPTYDEREIEAVVAALRSGELATGVTTRKFEAEFAESFGFAHALAVTSGSTANLLACAAMLELGRARPGDRVIVSGATFVSAVTPVVQLGLVPVFVDVAAGHVNVDLDLVEQAVVEHGARGVLLPHTLGQALPMDRLAEIKRRHGVFVIEDCCESLGAADGSTPVGSAADVATFSFYAGHHLTM GEGGVAAGHSAEIDSVLRSLAFGRNPDYRLGRFEHPVDDRPLAPEERYIHLLRGYNAKITDFQAAFGRVQLTRHAELARQRRQLAQELVPVLREFGWGVLGDPVSPGASPFAVATLLPEGLPLTRAVGVLIEHGIDPRGFLGASQPHQPCFDGVTKVVHEPYLHTRTLAERGLLLGCPPRTDRAAAVKALRRALESLS;

[0023] The amino acid sequence of the transaminase KsgC is shown in SEQ ID NO.5:

[0024] MSDSVLLSARQPLPPIDYSHAAGAWIHTAEGDSLLDAASGLVCVNIGHAHPHVVEQITRQARTATFASPGVLLPAVQEELAGRRLTEAVNRPGDGVSLACSGTSAVELAI SYARLIQRSRGQDGRHHILTARLGYHGNSALTLGLSGHRRRRPHPDDALGLAPAFDPPYPGHHRDCPHDRCRASCADAVAEAIDRRGPESVAAVLIEPVNGTTGGAYTP PPGYLAALRRACHERGVLVIHDEVLTGLGRTGLPLGADHCTDAAADIVVLSKGLSAGYLPLAAVLVNPEGVARLRSGPRPLPLMGTMSATPLQAAAGLAVLDVLGEIGA LDPARVRGGDVAAAVRAVAALPVVQETRGVGYFHAVELAPGTQAAALAAARDERLLLYPFNGFHADGTGEGLIVAPPLNSTPSEVEFLAQGLRRALERTAARSEDRSHR.

[0025] In this invention, the reaction buffer system is PBS buffer, and the cofactor of the buffer system includes Mg. 2+pyridoxal phosphate, glutamic acid or monosodium glutamate and glutamine;

[0026] In this invention, the reaction temperature is 26℃~30℃ and the reaction time is 2~4 hours.

[0027] This invention also provides the application of uridine-5′-diphosphate polydeoxydiaminosaccharide compounds or uridine-5′-diphosphate polydeoxydiaminosaccharide compounds prepared by the above preparation method in the preparation of kasugamycin and its derivatives.

[0028] The glycosidic unit of UDP-kasugamycin and its structural analog UDP-3”-hydroxy-kasugamycin provided by this invention is consistent with the core skeleton of kasugamycin, and can be used to prepare commercially available kasugamycin and its derivatives with no or low toxicity. The preparation method provided by this invention is simple to operate, operates under mild conditions, is environmentally friendly, and has a high product conversion rate, showing significant application prospects.

[0029] In this invention, the enzymatic synthesis routes of the compounds shown in Formulas 2 and 3 are as follows: Figure 1 As shown. Attached Figure Description

[0030] Figure 1 The enzymatic synthesis routes for the compounds shown in Formulas 2 and 3 are as follows;

[0031] Figure 2 SDS-PAGE analysis of five recombinant proteins;

[0032] Figure 3 High-resolution mass spectra of the compounds shown in Formulas 2 and 3;

[0033] Figure 4 The compound UDP-kasugamine shown in Formula 2 1 HNMR spectrum;

[0034] Figure 5 The compound UDP-kasugamine shown in Formula 2 1 C NMR spectrum;

[0035] Figure 6 The compound UDP-3”-hydroxy-kasugamine shown in Formula 3 1 H NMR spectrum;

[0036] Figure 7 The compound UDP-3”-hydroxy-kasugamine shown in Formula 3 1 C NMR spectrum;

[0037] Figure 8 The high-resolution mass spectra of the compound UDP-kasugamine shown in Formula 2 before and after labeling are shown.

[0038] Figure 9 High-resolution mass spectra before and after kasugamycin labeling. Detailed Implementation

[0039] For reagents or instruments used in the following text where specific technical or conditional specifications are not given, standard experimental conditions shall apply. If no reagent company instructions are explicitly provided, the conditions recommended in those instructions shall be followed. Reagents or instruments whose manufacturers are not specified are all commercially available standard products.

[0040] Example 1

[0041] Enzymatic preparation of UDP-kasugamine

[0042] (1) Construction of strains of enzymes for catalysis

[0043] Using the genome of *Streptomyces kasugamushi* CGMCC 4.1057 as a template, the target genes ksgQ (SEQ ID NO.1), ksgB (SEQ ID NO.2), ksgD (SEQ ID NO.3), ksgR (SEQ ID NO.4), and ksgC (SEQ ID NO.5) were amplified using specific primers. The specific primers are shown in Table 1.

[0044] Table 1 Primers used for amplifying the target gene.

[0045] *The restriction enzyme sites are marked with an underline.

[0046] The target gene was ligated between the NdeI and BamHI restriction sites of the expression vector pET-28a(+) using a one-step cloning method, resulting in five recombinant plasmids: pET28a-KsgQ, pET28a-KsgB, pET28a-KsgD, pET28a-KsgR, and pET28a-KsgC.

[0047] Five recombinant plasmids were transformed into E. coli BL21(DE3) competent cells to obtain five corresponding recombinant strains: 21 / pET28a-KsgQ, 21 / pET28a-KsgB, 21 / pET28a-KsgD, 21 / pET28a-KsgR, and 21 / pET28a-KsgC.

[0048] (2) Induced expression of recombinant proteins

[0049] The recombinant strains were streaked onto LB solid medium and incubated at 37°C until single colonies appeared. Each single colony was inoculated into 3 mL of LB liquid medium and incubated overnight at 37°C and 220 rpm to obtain seed cultures. The seed cultures were then transferred at a 1% (v / v) inoculation rate to 200 mL of LB liquid medium and incubated at 37°C and 220 rpm until OD (dose retardation) was achieved. 600 When the concentration reaches 0.6–0.8, add isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 0.1 mM for induction, and then incubate the shake flask at 16 °C and 180 rpm for 15–20 h.

[0050] (3) Recombinant protein purification and detection

[0051] The bacterial cells were collected by centrifugation, and the cell walls were disrupted using an ultrasonic cell disruptor. The disrupted cell lysate was centrifuged at 12,000 rpm for 40 min. The supernatant after centrifugation was purified using nickel ion affinity resin. During purification, the nickel column was first equilibrated with binding buffer, then impurities were eluted with wash buffer, followed by elution buffer to elute the target protein. Finally, the purified target protein was desalted and concentrated using a PD-10 desalting column and a 10 kDa ultrafiltration tube.

[0052] The purity of the recombinant target protein was detected using SDS-PAGE. The SDS-PAGE analysis results are as follows: Figure 2 As shown, the recombinases KsgQ (42.1 kDa), KsgB (41.5 kDa), KsgD (35.6 kDa), KsgR (42.6 kDa), and KsgC (45.4 kDa) were all successfully expressed, and the size of the target band was consistent with the theoretical molecular weight of the protein.

[0053] Protein concentration was determined using the BCA protein quantification kit.

[0054] LB solid medium: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride, 20 g / L agar powder, autoclaved at 121°C for 20 minutes.

[0055] LB liquid medium: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride, autoclaved at 121°C for 20 minutes.

[0056] Binding buffer: 20mM Tris-HCl (pH=7.4), 500mM sodium chloride, 5mM imidazole, 10% glycerol.

[0057] Washbuffer: 20mM Tris-HCl (pH=7.4), 500mM sodium chloride, 30mM imidazole, 10% glycerol.

[0058] Elution buffer: 20 mM Tris-HCl (pH = 7.4), 500 mM sodium chloride, 200 mM imidazole, 10% glycerol. The enzymatic synthesis reaction system for UDP-kasugamine includes:

[0059]

[0060] The buffer solution was PBS buffer (pH 7.4), and the reaction conditions were 28°C for 2 hours.

[0061] After the reaction was completed, the mixture was extracted three times with an equal volume of chloroform, and the upper aqueous phase was analyzed by HPLC.

[0062] (4) HPLC detection method for the product

[0063] Column: Dionex CarboPac TM PA1, 4×250mm

[0064] Mobile phase A: Water;

[0065] Mobile phase B: 1.0M ammonium acetate solution (adjusted to pH 8.0 with ammonia)

[0066] The elution procedure is as follows:

[0067]

[0068] Flow rate: 1.0 mL / min; column temperature: 30℃; detection wavelength: 260 nm; injection volume: 10 μL.

[0069] (5) Isolation and purification of UDP-kasugamine

[0070] The reaction system was expanded to 10 mL to obtain sufficient product for separation, purification, and structural identification. The reaction product was purified using techniques such as Sephadex LH-20 dextran gel and HPLC-Dionex CarboPac™ PA1 (semi-preparative) anion exchange, and then freeze-dried to obtain a high-purity product.

[0071] (6) Structural identification of UDP-kasugamine

[0072] The target compound, UDP-kasugamine, is a white powder solid. High-resolution mass spectrometry shows a quasi-molecular ion peak at m / z 531.0898 [MH]. - (See Figure 3 By precisely fitting the elemental composition, the molecular formula is deduced to be C.15 H 26 N4O 13 P2 (calculated value m / z 531.0899, [MH]) - The compound shown in Formula 2 1 H NMR spectrum (D2O, 500MHz) as follows Figure 4 As shown, 13 CNMR spectrum (D2O, 125MHz) as follows Figure 5 As shown in Table 2, the specific NMR data are assigned to different data points.

[0073] Table 2. NMR data assignment of product UDP-kasugamine

[0074] No. H (ppm, J = Hz) ​ C (ppm, J = Hz) ​ 1 2 151.7 3 4 166.2 5 5.95 (d, J = 8.1 Hz, 1 H) 102.5 6 7.94 (d, J = 8.1 Hz, 1 H) 141.6 1′ 5.97 (d, J = 4.4 Hz, 1 H) 88.5 2′ 4.35 (m, 1 H) 69.6 3′ 4.36 (m, 1 H) 73.7 4′ 4.29 (m, 1 H) 83.1 (d, J = 9.1 Hz) 5′ 4.16-4.26 (m, 2 H) 64.9 (d, J = 5.0 Hz) 1” 5.60 (d, J = 7.7 Hz, 1 H) 91.5 (d, J = 4.6 Hz) 2” 3.75 (m, 1 H) 48.4 (d, J = 10.5 Hz) 3” 2.30-2.45 (m, 2 H) 25.2 4” 3.40 (m, 1 H) 47.0 5” 4.32 (m, 1 H) 66.9 6” 1.37 (d, J = 6.3 Hz, 3 H) 16.6

[0075] In this embodiment, the HPLC purity of the prepared UDP-kasugamine product was 98%, and the total yield was 83%.

[0076] Example 2

[0077] Enzymatic preparation of UDP-3”-hydroxy-kasugamine

[0078] (1) Construction of strains of enzymes for catalysis

[0079] The specific solution is the same as in Implementation Example 1.

[0080] (2) Induced expression of recombinant proteins

[0081] The specific solution is the same as in Implementation Example 1.

[0082] (3) HPLC detection method for the product

[0083] The specific solution is the same as in Implementation Example 1.

[0084] (4) Recombinant protein purification and detection

[0085] The specific solution is the same as in Implementation Example 1.

[0086] The enzymatic synthesis reaction system for UDP-3”-hydroxy-kasugamine includes:

[0087]

[0088] The buffer solution was PBS buffer (pH 7.4), and the reaction conditions were 28°C for 2 hours.

[0089] After the reaction was completed, the mixture was extracted three times with an equal volume of chloroform, and the upper aqueous phase was analyzed by HPLC.

[0090] (5) Isolation and purification of UDP-3”-hydroxy-kasugamine

[0091] The specific solution is the same as in Implementation Example 1.

[0092] (6) Structural identification of UDP-3”-hydroxy-sphingamine

[0093] The target compound, UDP-3”-hydroxy-kasugamine, is a white powder solid. High-resolution mass spectrometry shows a quasi-molecular ion peak at m / z 547.0850 [MH]. - (See Figure 3 By precisely fitting the elemental composition, the molecular formula is deduced to be C. 15 H 26 N4O 14 P2 (calculated value m / z 547.0848, [MH]) - The compound shown in Formula 3 1 H NMR spectrum (D2O, 500MHz) as follows Figure 6 As shown, 13 CNMR spectrum 9 (D2O, 125MHz) Figure 7 As shown in Table 3, the specific NMR data are assigned to different data points.

[0094] Table 3. NMR data assignments for product UDP-3”-hydroxy-sphingamine

[0095] No. H (ppm, J = Hz) ​ C (ppm, J = Hz) ​ 1 2 151.8 3 4 166.2 5 5.96 (d, J = 8.1 Hz, 1 H) 102.6 6 7.96 (d, J = 8.1 Hz, 1 H) 141.6 1′ 5.98 (d, J = 4.4 Hz, 1 H) 88.4 2′ 4.36 (m, 1 H) 69.6 3′ 4.37 (m, 1 H) 73.8 4′ 4.29 (m, 1 H) 83.1 (d, J = 9.0 Hz) 5′ 4.20-4.24 (m, 2 H), 64.9 (d, J = 5.0 Hz) 1” 5.76 (d, J = 7.9 Hz, 1 H) 92.5 (d, J = 4.7 Hz) 2” 3.85 (br d, 1 H) 53.3 (d, J = 10.4 Hz) 3” 4.47 (dd, J = 10.6, 4.7 Hz, 1 H) 62.9 4” 3.21 (t, J = 10.6 Hz, 1 H) 53.2 5” 4.35 (m, 1 H) 65.9 6” 1.41 (d, J = 6.3 Hz, 3 H) 16.7

[0096] In this embodiment, the HPLC purity of the prepared UDP-3”-hydroxy-kasugamine product was 95%, and the total yield was 72%.

[0097] Application Example 1

[0098] UDP-kasugamycin is an intermediate in the production of the commercial antibiotic kasugamycin.

[0099] (1) 15 Preparation of N-UDP-Springmine

[0100] This application example utilizes L-[α- 15 [N]-glutamic acid was used to prepare the UDP-kasugamine from Example 1 by isotopic labeling at the 4-NH2 position. 15 N-UDP-Springmine, specifically:

[0101] 15 The enzymatic synthesis reaction system for N-UDP-kasugamine includes:

[0102]

[0103] 15 The separation and purification scheme for N-UDP-kasugamine is the same as in Example 1.

[0104] like Figure 8As shown, high-resolution mass spectrometry reveals the prepared... 15 The quasi-molecular ion peak of N-UDP-kasugamine is 532.0861 [MH]. - (Calculated value m / z 532.0869, [MH]) - The molecular weight of UDP-kasugamine increased by 1 Da compared to the unlabeled UDP-kasugamine, indicating that... 15 N-UDP-kasugamine labeling was successful.

[0105] (2) Isotopic labeling of kasugamycin

[0106] Will get 15 N-UDP-kasugamycin was fed to wild-type kasugamycin-producing strain *Streptomyces kasugamushi* CGMCC 4.1057, specifically as follows:

[0107] Wild-type Streptomyces kasugamushi CGMCC 4.1057, grown for 4 days, was cell-disrupted using an ultrasonic cell disruptor. The cell lysate was centrifuged at 12,000 rpm for 40 min, and the supernatant obtained was the total protein extract of Streptomyces kasugamushi CGMCC 4.1057.

[0108] The final concentration was 0.1 mM. 15 N-UDP-kasugamine was added to the total protein extract of Streptomyces kasugatum CGMCC 4.1057.

[0109] The buffer solution was PBS buffer (pH 7.4), and the reaction conditions were 28°C for 4 hours.

[0110] After the reaction was completed, the mixture was extracted 5-10 times with an equal volume of chloroform. The upper aqueous phases were combined, lyophilized, resuspended in deionized water, and analyzed by high-resolution mass spectrometry.

[0111] like Figure 9 As shown, high-resolution mass spectrometry displays 15 N-UDP-kasugamine was incorporated into the target product kasugamycin, resulting in a significant increase in the isotope peak of kasugamycin.

[0112] This application example demonstrates that UDP-kasugamycin is an intermediate compound in the biosynthesis of kasugamycin, and it has significant application prospects in the preparation of commercially available agricultural and medical antibiotics such as kasugamycin and its derivatives with no or low toxicity.

[0113] The above description is merely a preferred embodiment of the present invention and should not be construed as limiting the scope of the invention. Any variations and improvements made to the present invention should be included within the scope of protection of the present invention.

Claims

1. A polydeoxydiaminosaccharide compound of uridine-5'-bisphosphate, characterized in that, The compounds include the structures, pharmaceutically / pesticide-acceptable salts, esters, and prodrugs as described in Formula 1; The R includes H and OH; When R is H, it is named UDP-Spring Thunderamine, and the structure is shown in Formula 2: When R is OH, it is named UDP-3”-hydroxy-kasugamine, and the structure is shown in Formula 3:

2. The method for preparing the compound according to claim 1, characterized in that, When preparing UDP-kasugamine, the preparation method includes the following steps: using uridine-5'-bisphosphate-N-acetyl-glucosamine as a substrate, a one-pot multi-enzyme reaction is carried out in a buffer system by isomerase KsgQ, deacetylase KsgB, dehydratase KsgD, dehydrogenase KsgR and transaminase KsgC, and the product is obtained after purification; When preparing UDP-3”-hydroxy-kasugamine, the preparation method includes the following steps: using uridine-5'-bisphosphate-N-acetyl-glucosamine as a substrate, a one-pot multi-enzyme reaction is carried out in a buffer system by isomerase KsgQ, deacetylase KsgB, dehydratase KsgD and transaminase KsgC, and the product is obtained after purification.

3. The preparation method according to claim 2, characterized in that, The amino acid sequence of the isomerase KsgQ is shown in SEQ ID NO.1; the amino acid sequence of the deacetylasase KsgB is shown in SEQ ID NO.2; the amino acid sequence of the dehydratase KsgD is shown in SEQ ID NO.3; the amino acid sequence of the deoxygenase KsgR is shown in SEQ ID NO.4; and the amino acid sequence of the transaminase KsgC is shown in SEQ ID NO.

5.

4. The preparation method according to claim 2, characterized in that, The reaction buffer system is PBS buffer, and the cofactor of the buffer system includes Mg. 2+ Pyridoxal phosphate, glutamic acid or sodium glutamate and glutamine.

5. The preparation method according to claim 2, characterized in that, The reaction temperature is 26℃~30℃, and the reaction time is 2~4 hours.

6. The use of the compound of claim 1 or the compound prepared by any one of the preparation methods of claims 2 to 5 in the preparation of kasugamycin and its derivatives.

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