Rhamnosyltransferase sgt822 and use thereof

Abstract

The application discloses a rhamnosyltransferase SGT822 and application thereof. The applicant screens a rhamnosyltransferase SGT822 from Streptomyces sp. 147326, which can glycosylate and modify nosiheptide to produce rhamnosylated nosiheptide, greatly improves the water solubility of the nosiheptide, explains the modifiability of a hydroxyl group of a pyridine ring of NOS, provides a new site selection for structural modification of NOS, and provides a new enzymatic element for rhamnosylated modification of natural products.

Description

Technical Field

[0001] This invention relates to a rhamnosyltransferase, specifically to rhamnosyltransferase SGT822 and its applications. Background Technology

[0002] Thiopeptide antibiotics belong to the ribosome synthesis and posttranslational modified peptides (Ripps), and are a class of highly modified heterocyclic peptide antibiotics produced by various bacteria. They consist of three parts: a central pyridine ring, a core macrocycle, and a tail (Bagley MC, et al. J. Chem Rev. 2005 Feb; 105(2):685-714).

[0003] The structure of nusiopeptide is as follows:

[0004]

[0005] Nosiheptide (NOS) belongs to the e-series of thiopeptides and was initially isolated from Streptomyces cerevisiae. It exhibits excellent antibacterial activity against all methicillin-resistant and methicillin-sensitive Staphylococcus aureus (MRSA) strains. Due to its representative structure, it has become a mainstream antibiotic in thiopeptide research (Haste NM, et al. J. Antibiot (Tokyo) 2012 Dec; 65(12):593-8.). Although NOS has strong antibacterial activity, its poor water solubility and inactivation in vivo have limited its drug development and clinical application. Structural modification to obtain structurally diverse analogs has become one of the important means to improve the drugability of NOS. However, the structure of NOS is too complex and extremely difficult to modify. There are currently few successful examples of modification, and most of them focus on the modification of the dehydroalanine side chain and side ring.

[0006] In the structure of NOS, the core pyridine ring hydroxyl group has excellent reactivity and is an ideal site for structural modification. Rhamnoseptide pyridine ring hydroxyl group is glycosylated using rhamnoseptide to form rhamnoseptide, thus enhancing its water solubility. Summary of the Invention

[0007] The purpose of this invention is to provide a rhamnosyltransferase SGT822 and its applications. Rhamnosyltransferase SGT822 can achieve regioselective glycosylation modification of the NOS pyridine ring hydroxyl group. To achieve the above objective, the technical solution adopted by this invention is as follows:

[0008] 1) The rhamnosyltransferase is SGT822 derived from Streptomyces sp. 147326.

[0009] 2) Rhamnosyltransferase SGT822, the nucleotide sequence of which is as described in SEQ ID NO: 1, efficiently catalyzes the formation of rhamnosyltransferase from nosicin to rhamnosicin.

[0010] 3) Construct plasmids from rhamnosyltransferase SGT822.

[0011] 4) Rhamnosyltransferase SGT822 can be expressed independently or co-expressed in E.coli BL21(DE3). This heterologous expression host is only an example and does not limit the scope of the present invention. Rhamnosyltransferase can also be expressed in lactic acid bacteria, streptomyces, yeast, Bacillus subtilis, etc.

[0012] 5) This invention provides a rhamnosyltransferase that has the reactivity to catalyze the conversion of nosity peptide to rhamnosity peptide.

[0013]

[0014] The present invention has the following advantages:

[0015] This invention discovers that the rhamnosyltransferase SGT822, derived from natural microorganisms, is a novel enzyme with a structure not reported in the literature. It has the function of selectively modifying the pyridine ring hydroxyl group of nositide to form rhamnositide, that is, it can perform site-specific modification. In other words, since NOS has two hydroxyl groups, this invention can selectively glycosylate rhamnosyltransferase in the region of the pyridine ring hydroxyl group of NOS, while the other threonine 3-position hydroxyl group remains unaffected.

[0016] This improved the water solubility of nusiti, providing a new approach for other compounds in this series to utilize this site for enhanced water solubility. Attached Figure Description

[0017] Figure 1 HPLC detection of NOS catalyzed by SGT822;

[0018] Figure 2 To investigate the effect of pH on SGT822;

[0019] Figure 3 The effect of reaction temperature on SGT822;

[0020] Figure 4 The effects of temperature stability (A) and pH stability (B) on SGT822;

[0021] Figure 5 The effect of metal ions on SGT822;

[0022] Figure 6Michaelis equation curves for the generation of rhamnosylated nosikeptide from SGT822 glycosylation modification of nosikeptide;

[0023] Figure 7 Elution curve of SGT822 after passing through Superdex 200;

[0024] Figure 8 This is a schematic diagram of the pET22b-SGT822 plasmid. Detailed Implementation

[0025] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0026] Example 1: Construction of pET22b-SGT822 expression plasmid

[0027] (a) Acquisition of target genes

[0028] The genome of Streptomyces sp. 147326 was obtained using a kit extraction method. Specific primers were designed as shown in Table 1. The DNA fragment of the target sequence SGT822 was obtained by PCR amplification and purified by gel extraction.

[0029] Table 1. SGT822-specific primers

[0030]

[0031] (b) Enzyme digestion of the target fragment and the vector

[0032] The target fragment and vector were digested using restriction endonucleases, and the digestion system is shown in Table 2. After mixing, the digestion system was incubated at 37°C for 1 h. The digestion products were separated by 1% agarose gel electrophoresis at 120 V for 30 min. The digested fragments were recovered using a gel recovery kit.

[0033] Table 2. Enzyme digestion system

[0034]

[0035] (c) Target fragment and vector ligation

[0036] The target fragment recovered after enzyme digestion was ligated to the linear vector. The ligation system is shown in Table 3. After mixing, the system was incubated at 16°C for 12 hours. A schematic diagram of the pET22b-SGT822 plasmid is shown below. Figure 8 As shown.

[0037] Table 3. Target fragment to vector linkage system

[0038]

[0039] Example 2: Protein expression and purification of pET22b-SGT822 plasmid

[0040] (a) Construction of expression strain

[0041] The pET22b-SGT822 plasmid was transformed into E. coli competent cells DH5α, and sequencing yielded the correct plasmid pET22b-SGT822. The pET22b-SGT822 plasmid was then transformed into E. coli protein expression competent cells.

[0042] BL21(DE3).

[0043] (b) Induced expression of rhamnosyltransferase SGT822

[0044] Single clones of the strain were selected and cultured in liquid LB medium at 37°C with shaking at 220 rpm until OD reached. 600 =0.6,

[0045] IPTG was added to a final concentration of 0.5 mM, and the mixture was induced at 20°C for 12 h. After induction, the bacterial cells were collected.

[0046] The bacterial cells were resuspended in 50 mM Tris-HCl and collected by centrifugation again.

[0047] (c) Purification of rhamnosyltransferase SGT822

[0048] Weigh 5g of bacterial cells and add 50mL of Tris-HCl buffer, mix well. Disrupt the cells using an ultrasonic cell disruptor, collect the supernatant, and purify it using nickel affinity chromatography to obtain SGT822 rhamnosyltransferase.

[0049] Example 4: Functional Verification of Rhamnosyltransferase SGT822

[0050] The function of rhamnosyltransferase SGT822 was verified according to the reaction system shown in Table 4, with NOS as the acceptor and dTDP-L-rhamnose (dTR) as the sugar donor. After reacting at 30℃ and 100 rpm for 6 h, 500 μL of methanol and 500 μL of DMSO were added and mixed to terminate the reaction. The mixture was centrifuged at 12000 rpm for 10 min, filtered through a 0.22 μm organic filter, and analyzed by HPLC using pure NOS-R as a positive control. The results are shown below. Figure 1 As shown.

[0051] Table 4. Glycosyltransferase reaction system

[0052]

[0053] Example 5 Enzymatic Study of Rhamnosyltransferase SGT822 (a) Determination of Enzymatic Properties

[0054] 1. Optimal reaction pH

[0055] The reaction system was set to a final concentration of 1 mg / mL NOS, 20 μL of SGT822 obtained in Example 3, and 100 μL of dTR. Three parallel experiments were performed for each sample. After the reaction, HPLC analysis was performed to examine the change in substrate conversion within the pH range of 6–11. Figure 2 The results showed that when the reaction pH was between 6 and 10, the yield increased with increasing pH, reaching a maximum conversion of 39.1% at pH 10, using a 50 mM Glycine-NaOH buffer. Furthermore, different buffers used at the same pH also affected enzyme activity, with Tris having the least impact.

[0056] 2. Optimal reaction temperature

[0057] The change in substrate conversion was investigated within the temperature range of 25–70 °C, using the same reaction system as above. Figure 3 The results showed that when the reaction temperature was between 25 and 55°C, the yield increased with increasing temperature, and the conversion rate reached its highest at 55°C, at 73.2%. As the temperature continued to rise, the yield decreased significantly, reaching only 13.3% at 70°C.

[0058] 3. Temperature stability and pH stability

[0059] like Figure 4 The results showed that SGT822 exhibited good stability at 4°C, with no significant change in enzyme activity after 12 hours of storage. However, its stability at 30°C was worse than that at 4°C. After storage in 50mM Glycine-NaOH buffer for 12 hours, the enzyme activity decreased by only 7%, indicating that it is suitable as a storage buffer for SGT822.

[0060] 4. Influence of metal ions

[0061] Add EDTA and Mg to a final concentration of 5 mM 2+ Mn 2+ Zn 2+ Fe 3+ Cu 2+ Ca 2+ K + Na + and Ni 2+ The remaining reaction system is the same as above. The reaction is carried out at the enzyme's optimal temperature and pH. The relative activity is calculated using the substrate conversion rate of the control group without metal ions as the standard. Figure 5 As shown, Mg2+ It increased enzyme activity by approximately 1.3 times, while the metal ion chelating agent EDTA decreased enzyme activity by half. Other metal ions all had varying degrees of inhibitory effects on SGT822, Mn 2+ Ca 2+ K + and Na + The effect on enzyme activity is relatively weak, while Cu 2+ The inhibitory effect was the strongest, causing the enzyme activity to be lost by about 97%. (b) Determination of kinetic parameters of rhamnosyltransferase SGT822.

[0062] The kinetic parameters of a single substrate, NOS, were determined. The dTR was fixed at 100 μL, and the final NOS concentration was set between 0.0156 and 2 mM. The reaction was carried out for 45 min at the optimal temperature and pH. The nonlinear fitting curve is shown below. Figure 6 As shown, the maximum reaction rate Vmax was 1.465 ± 0.049 μM / min, the Michaelis constant Km was 107.22 ± 13.38 μM, and the catalytic constant Kcat was 0.033 ± 0.001 min. -1 And Kcat / Km is 3.07×10 -3 min -1 μM -1 .

[0063] Example 6: Active form of rhamnosyltransferase SGT822

[0064] Gel chromatography was used to separate monomers and oligomers with significantly different molecular weights; proteins with larger molecular weights eluted before those with smaller molecular weights. The elution results are as follows: Figure 7 As shown, two peaks were observed at 280 nm. Using the standard curve of the control protein, the first peak represented the multimer, and the second peak represented the monomer. Activity verification was performed after collecting the monomers and multimers separately. The results showed that the monomers were active, while the multimers were inactive. Example 7: Rhamnosyltransferase SGT822 catalyzes residue localization.

[0065] Conserved residues His13 and Asp308 were selected as mutation sites, and they were mutated to alanine using a site-directed mutagenesis kit. The primers required for this process are shown in Table 5 (bold bases indicate mutation sites), and the PCR program is shown in Table 6. As shown in the following formula, His13 and Asp308 are indispensable for catalysis, acting as a catalytic duo to exert activity.

[0066]

[0067] Table 5. Primers used for alanine mutation

[0068]

[0069] Table 6. PCR Amplification Procedure

[0070]

Claims

1. A rhamnosyltransferase SGT822, characterized in that, The amino acid sequence of the rhamnosyltransferase SGT822 is shown in SEQ ID NO.

2.

2. A recombinant expression vector, characterized in that, The recombinant expression vector contains a nucleotide sequence encoding the rhamnosyltransferase SGT822 gene of claim 1, as shown in SEQ ID NO.

1.

3. An engineered host cell, characterized in that, The engineered host cell comprises the recombinant expression vector of claim 2.

4. The engineered host cell according to claim 3, characterized in that, The host cell is Escherichia coli.

5. The application of the rhamnosyltransferase SGT822 according to claim 1 or the host cell according to claim 3 or 4 in the synthesis of rhamnosylated nosity peptide from glycosylated nosity peptide.

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