A tylosin polyketide synthase acyl transferase mutant and use thereof
By site-directed mutagenesis and recombinant vector construction of tylosin polyketide synthase acyltransferase, the substrate specificity and catalytic efficiency of tylosin polyketide synthase acyltransferase were improved, solving the problems of low substrate specificity modification and low catalytic efficiency in the existing technology, and obtaining a highly efficient tylosin polyketide synthase acyltransferase mutant.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-03-23
- Publication Date
- 2026-03-27
AI Technical Summary
Existing technologies have not yet modified the substrate specificity of tylosin polyketide synthase acyltransferase, and the catalytic efficiency is low.
By performing site-directed mutagenesis on the key amino acid sequence of tylosin polyketide synthase acyltransferase, especially modifying positions 335, 337, 263, and 388, a recombinant vector was constructed and the protein was expressed and purified, thereby improving the efficiency of malonyl-CoA catalysis.
Substrate-specific modification of tylosin polyketide synthase acyltransferase was achieved, increasing the efficiency of malonyl-CoA catalysis by 2.6 times. A highly efficient tylosin polyketide synthase acyltransferase mutant was obtained for activity assay experiments.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of bioengineering, and particularly relates to a tylosin polyketide synthase acyltransferase mutant and application thereof. BACKGROUND
[0002] Polyketides are a class of secondary metabolites produced by bacteria, actinomycetes, fungi or plants, including macrolides, tetracyclines, anthracyclines, polyethers, etc., which have antifungal, anti-infective, antitumor, immunosuppressive and other activities. Polyketides are synthesized by polyketide synthases, which can be divided into type I PKS, type II PKS and type III PKS. Type I PKS is composed of different modules, each module includes a set of domains to extend or modify the polyketide chain. The acyltransferase domain, ketosynthase domain and acyl carrier protein domain constitute three core domains, and each enzyme domain plays a different role in the extension and modification of the growing polyketide.
[0003] Acyltransferase (AT) catalyzes the formation of AT-acyl intermediate and the release of coenzyme A from acyl substrate, and the AT-acyl intermediate transfers the acyl group to acyl carrier protein (ACP). The histidine in the active center of AT deprotonates the catalytic amino acid serine, enhances the nucleophilicity of serine, and promotes serine to attack the carbonyl carbon of the substrate to form AT-acyl intermediate. Subsequently, the AT-acyl intermediate attacks the thiol of the phosphopantetheine arm on the ACP to transfer the acyl group to the ACP. The ketosynthase (KS) domain catalyzes the extension of the polyketide chain by two carbon atoms through a Claisen condensation.
[0004] The acyltransferase domain in modular polyketide synthases naturally has specificity for several different α-carboxyl-CoA substrates, the most common substrates being malonyl-CoA and methylmalonyl-CoA, with ethylmalonyl-CoA being less common. Previous studies have shown that the final product obtained by acyltransferase recognizing different substrates will have different biological activities. The acyltransferase of module 3 of epothilone polyketide synthase can recognize and utilize two substrates, malonyl-CoA and methylmalonyl-CoA, to generate two different main products, epothilone A and epothilone B, among which epothilone B has stronger antitumor activity. The acyltransferase of module 8 of polymyxin polyketide synthase can also recognize two substrates to generate two main products, polymyxin A and polymyxin D, among which polymyxin A has stronger insecticidal activity. Therefore, it is of great significance to obtain polyketides with stronger activity by modifying the substrate specificity of polyketide synthase acyltransferase.
[0005] Currently, there have been some reports on the modification of acyltransferases. Multiple sequence alignment and crystal structure studies have identified several key amino acid residues in the active site of acyltransferases and its vicinity. Site-directed mutagenesis of important residues allows the acyltransferase domain to remain in its natural environment and minimizes interference with important protein-protein interactions. After analyzing the amino acid sequence of acyltransferases, it is found that the position about 100 amino acids from the C-terminus of the protein is the active center of acyltransferases, and the amino acid sequence of the active center determines the substrate specificity of acyltransferases. The substrates recognized by acyltransferases can be inferred from the conserved motifs in the active center. The active center of acyltransferases recognizing malonyl-CoA usually contains the motif HAFH, and the active center of acyltransferases recognizing methylmalonyl-CoA contains the motif YASH. Changing only the conserved sequence of acyltransferases can modify the substrate specificity, but the catalytic efficiency is usually low. There is no modification of the substrate specificity of tylosin polyketide synthase acyltransferase in the prior art.
[0006] Therefore, the skilled in the art is committed to developing a mutant and application method for modifying the substrate specificity and improving the catalytic efficiency of tylosin polyketide synthase acyltransferase. SUMMARY
[0007] In view of the above defects of the prior art, the technical problem to be solved by the present application is to develop a mutant and application method for modifying the substrate specificity and improving the catalytic efficiency of tylosin polyketide synthase acyltransferase.
[0008] To achieve the above-mentioned object, the present application provides a tylosin polyketide synthase acyltransferase mutant, which includes a first mutant, a second mutant or a third mutant; the amino acid sequence of the first mutant is shown in SEQ ID NO. 2, the amino acid sequence of the second mutant is shown in SEQ ID NO. 3, and the amino acid sequence of the third mutant is shown in SEQ ID NO. 4.
[0009] Further, the first mutant of tylosin polyketide synthase acyltransferase is obtained by simultaneously mutating the 335th and 337th positions of the amino acid sequence shown in SEQ ID NO. 1; the second mutant is obtained by simultaneously mutating the 263rd, 335th and 337th positions of the amino acid sequence shown in SEQ ID NO. 1; and the third mutant is obtained by simultaneously mutating the 263rd, 335th, 337th and 388th positions of the amino acid sequence shown in SEQ ID NO. 1.
[0010] Further, the threonine at position 335 and the glycine at position 337 in the amino acid sequence shown in SEQ ID NO. 1 are mutated into histidine and phenylalanine respectively (T335H / G337F), to obtain a tylosin polyketide synthase acyltransferase first mutant; the tryptophan at position 263 in the tylosin polyketide synthase acyltransferase first mutant is mutated into methionine (T335H / G337F / W263M), to obtain a tylosin polyketide synthase acyltransferase second mutant; and the methionine at position 388 in the tylosin polyketide synthase acyltransferase second mutant is mutated into alanine (T335H / G337F / W263M / M388A), to obtain a tylosin polyketide synthase acyltransferase third mutant.
[0011] Further, based on the structure and sequence alignment, the key amino acids in the active center are selected for site-directed mutation, and the acyltransferase domain TyIAT5 of the tylosin polyketide synthase module five is successfully modified to modify the substrate specificity.
[0012] The application also provides a gene encoding the tylosin polyketide synthase acyltransferase mutant.
[0013] The application also provides a recombinant vector constructed by the gene encoding the tylosin polyketide synthase acyltransferase mutant.
[0014] Further, the gene fragment range of the acyltransferase domain TyIAT5 of the tylosin polyketide synthase module five is determined from the Streptomyces fradiae genome based on the boundary determination of the tylosin polyketide synthase acyltransferase domain, and a recombinant expression vector containing the gene fragment is constructed.
[0015] Further, based on the sequence alignment, the complete nucleic acid sequence encoding the acyltransferase domain and the connecting fragment between the ketosynthase and the acyltransferase is amplified by designing primers, and is connected to an expression vector to obtain an expression vector containing the target gene, which can be used for TylAT5 protein expression and purification.
[0016] The application also provides a recombinant bacterium prepared by transforming the recombinant vector constructed by the gene encoding the tylosin polyketide synthase acyltransferase mutant.
[0017] The application also provides a soluble expression method of the tylosin polyketide synthase acyltransferase domain TylAT5 protein, comprising the following steps:
[0018] Step 1, the recombinant bacterium is cultured in LB liquid medium at 37℃ for 6h; then the bacterium is transferred into LB liquid medium and cultured at 37℃ until the OD600 is 0.4-0.6, then IPTG is added and the culture is induced at 16℃ overnight; and the bacterium is collected by centrifugation at room temperature;
[0019] Step 2, the bacteria collected in step 1 are resuspended with buffer, broken by ice bath ultrasonic, centrifuged at 10000 rpm for 40 min at 4 DEG C, and the supernatant is taken; the target protein with a histidine tag is purified by nickel column affinity chromatography, the supernatant is flowed through twice, and the impure protein is eluted with the first imidazole buffer, and the target protein is eluted with the second imidazole buffer, to obtain an eluted solution;
[0020] Step 3, the eluted solution obtained in step 2 is centrifuged at 4 DEG C, and the precipitate is collected and further purified by fast protein liquid chromatography.
[0021] Further, the LB liquid medium in step 1 contains 50 mg / mL kanamycin; the final concentration of IPTG added in step 2 is 0.3 mM, the buffer composition is 50 mM Tris 500 mM NaCl, the composition of the first imidazole buffer is 50 mM Tris 500 mM NaCl 20 mM imidazole, and the composition of the second imidazole buffer is 50 mM Tris 500 mM NaCl 300 mM imidazole; the centrifugal speed in step 3 is 12000 rpm, and step 3 further comprises: using a superdex 200 10 / 300 chromatographic column, and using 10 mM Tris 150 mM NaCl pH 7.5 as the mobile phase.
[0022] Further, the optimal conditions for soluble expression of TylAT5 are explored, and a series of experimental procedures and conditions such as the culture temperature and time of the recombinant bacteria, the purification method, and the reagents used are determined, so as to obtain the acyltransferase domain TylAT5 protein of the tylosin polyketide synthase module five which is good in solubility, uniform in state, and monomeric. The TylAT5 protein is high in expression amount and good in state, and can be used for subsequent activity determination experiments.
[0023] The application further provides an application of a tylosin polyketide synthase acyltransferase mutant in catalyzing malonyl-CoA hydrolysis.
[0024] The application further provides an application of a coding gene of a tylosin polyketide synthase acyltransferase mutant in catalyzing malonyl-CoA hydrolysis.
[0025] The application further provides an application of a recombinant vector containing a tylosin polyketide synthase acyltransferase mutant in catalyzing malonyl-CoA hydrolysis.
[0026] The application further provides an application of a recombinant bacteria containing a tylosin polyketide synthase acyltransferase mutant in catalyzing malonyl-CoA hydrolysis.
[0027] In the preferred embodiment 1 of the present application, the construction process of the tylosin polyketide synthase acyltransferase recombinant bacteria is described in detail;
[0028] In another preferred embodiment 2 of the present application, the construction process of the tylosin polyketide synthase acyltransferase two-point mutation is described in detail;
[0029] In another preferred embodiment 3 of the present application, the construction process of the tylosin polyketide synthase acyltransferase three-point mutation is described in detail;
[0030] In another preferred embodiment 4 of the present application, the construction process of the tylosin polyketide synthase acyltransferase four-point mutation is described in detail;
[0031] In another preferred embodiment 5 of the present application, the induced expression of the tylosin polyketide synthase acyltransferase wild type and mutants is described in detail;
[0032] In another preferred embodiment 6 of the present application, the determination process of the kinetic parameters of the tylosin polyketide synthase acyltransferase wild type and mutants is described in detail.
[0033] The beneficial effects of the present application are as follows:
[0034] The present application reengineers the substrate specificity of tylosin polyketide synthase acyltransferase, determines the gene fragment range of the acyltransferase domain TylAT5 of the tylosin polyketide synthase module five, constructs a recombinant expression vector containing the gene fragment, successfully reengineers the substrate specificity, and expresses and purifies the protein of the tylosin polyketide synthase acyltransferase domain. The efficiency of catalyzing malonyl-CoA is improved, and four-point, three-point and two-point mutants with 2.6 times, 2.4 times and 1.85 times the activity of recognizing malonyl-CoA of the wild type are obtained. An expression vector containing the target gene is obtained, which can be used for TylAT5 protein expression and purification. A TylAT5 protein with high expression amount and good state is obtained and used for activity determination experiment. The present application provides a recombinant Escherichia coli for efficiently expressing acyltransferase mutants. Compared with the wild type acyltransferase, the efficiency of the mutant catalyzing malonyl-CoA is 2.6 times that of the wild type, which is 0.13±0.024 min-1μM-1. This provides a way for molecularly reengineering tylosin polyketide synthase acyltransferase.
[0035] The concept, specific structure and generated technical effects of the present application will be further described below with reference to the accompanying drawings, so as to fully understand the purpose, features and effects of the present application. BRIEF DESCRIPTION OF DRAWINGS
[0036] Figure 1 is a nucleic acid electrophoresis map of the tylAT5 gene fragment of a preferred embodiment 1 of the present application;
[0037] Figure 2 is a wild type SDS-PAGE electrophoretogram of recombinant bacteria of a preferred embodiment 5 of the present application;
[0038] Figure 3 is an enzymatic reaction kinetics curve of acyltransferase TylAT5 wild type and mutants under different substrate concentrations of a preferred embodiment 6 of the present application;
[0039] Figure 4 is a column chart of transacylase activity of acyltransferase TylAT5 wild type and mutants on malonyl-CoA of a preferred embodiment 6 of the present application. DETAILED DESCRIPTION
[0040] The following reference drawings of the specification introduce a plurality of preferred embodiments of the present application, so that the technical content thereof is more clear and convenient to understand. The present application can be embodied in many different forms of embodiments, and the protection scope of the present application is not limited to the embodiments mentioned herein. Unless otherwise specified, the various reagents and raw materials used in the present application are commercially available products or products that can be prepared by known methods.
[0041] Example 1: Construction of Acyltransferase Recombinant Bacteria
[0042] The acyltransferase from Streptomyces fradiae was selected, and the tylAT5 gene was amplified by PCR using the genome of Streptomyces fradiae as a template and tylAT5-F: 5'-ATCGTAATCCATATGtggcccgccggcccgtccgcg-3' (SEQ ID NO. 5) and tylAT5-R: 5'-TGATTCGATGAATTCActggaacgggtacgtcggcgggtt-3' (SEQ ID NO. 6) as primers, and Nde I and Eco RI restriction enzyme sites were introduced at the 5' and 3' ends of the tylAT5 gene, respectively. The PCR reaction system (50 μL) was as follows: 2 × Phanta Max Buffer 25 μL, template DNA 1 μL, upper and lower primers 2 μL each, dNTPs 1 μL, DMSO 2.5 μL, Phanta Max Super-Fidelity DNA Polymerase 1 μL, and sterile water 16.5 μL. The PCR reaction conditions were as follows: 95 °C pre-denaturation for 3 min; 95 °C denaturation for 30 s, 62 °C annealing for 15 s, 72 °C extension for 1.5 min, 25 cycles; 72 °C extension for 10 min. The PCR amplification product was verified by 1% agarose gel electrophoresis, and when the band was about 1.5 kb, which was consistent with the size of SEQ ID NO. 1, the product was recovered by a column-type DNA gel recovery kit. The recovered DNA and the plasmid pET28a were subjected to double enzyme digestion with restriction enzymes Nde I and Eco RI, respectively. After the enzyme digestion, the PCR amplification fragment was recovered by a column-type PCR product purification kit, and the plasmid digestion product was recovered by a column-type DNA gel recovery kit. The nucleic acid electrophoretogram of the tylAT5 gene fragment is shown in FIG. 1, wherein the left lane is a DNA Marker, and the right lane is the PCR amplification product of the tylAT5 gene. Figure 1 The recovered fragment and the vector were ligated at a molar ratio of 4:11, and the E. coli DH10B competent cells were transformed. The single colonies that grew were picked and expanded, and the plasmid pET28a-tylAT5 was extracted by a plasmid extraction kit. After sequencing, the E. coli BL21 (DE3) competent cells were transformed to obtain the recombinant bacteria E. coli BL21 (DE3) / pET28a-tylAT5.
[0043] Example 2: Construction of two-point mutation of acyltransferase
[0044] The recombinant plasmid pET28a-tylAT5 in Example 1 was subjected to homologous alignment with the amino acid sequences of TylAT3 and TylAT7, and the corresponding mutation sites in TylAT5 were found, and the mutation sites T335H, G337F, W263M and M388A were obtained. First, the conserved motif of the active center was mutated to obtain TylAT5 T335H / G337F. The site-directed mutation was completed by whole plasmid PCR. The template pET28a-tylAT5 was subjected to whole plasmid PCR with TylAT5 T335H / G337F-F: 5'-agccgatccccggcgtggaccacgccttccactccgcgcaggtggac-3'(SEQ ID NO. 7), TylAT5 T335H / G337F-R: 5'-gtccacgccggggatcggcttggcccggatgccctccg-3'(SEQ ID NO. 8) as primers, the template was digested with Dpnl, the PCR product was recovered with a column type PCR product purification kit, and E. coli DH10B competent cells were transformed. The single colonies grown were picked and expanded, the plasmid pET28a-tylAT5 T335H / G337F was extracted with a plasmid extraction kit, and after sequencing, E. coli BL21(DE3) competent cells were transformed to obtain the recombinant bacteria E. coli BL21(DE3) / pET28a-tylAT5 T335H / G337F. The two-point mutant amino acid sequence is SEQ ID NO. 2.
[0045] Example 3: Construction of three-point mutant of acyltransferase
[0046] The recombinant plasmid pET28a-tylAT5 T335H / G337F in Example 2 was used as a template for site-directed mutagenesis at position 263. The site-directed mutagenesis was completed by whole plasmid PCR. Primer TylAT5-W263M-F: 5'-tggcgctgcgcagccgggcaatgctcacactggccggcaa-3' (SEQ ID NO. 9); TylAT5-W263M-R: 5'-tgcccggctgcgcagcgccacgatccgcgccgcgtcct-3' (SEQ ID NO. 10). After the completion of PCR, the template was digested with Dpnl, the PCR product was recovered by a column PCR product purification kit, and E. coli DH10B competent cells were transformed. The single colonies that grew were expanded in culture, and the plasmid pET28a-tylAT5 T335H / G337F / W263M was extracted using a plasmid extraction kit. After sequencing, E. coli BL21(DE3) competent cells were transformed, and the recombinant bacteria E. coli BL21(DE3) / pET28a-tylAT5 T335H / G337F / W263M were obtained. The three-point mutant amino acid sequence was SEQ ID NO. 3.
[0047] Example 4: Construction of four-point mutant of acyltransferase
[0048] The recombinant plasmid pET28a-tylAT5 T335H / G337F / W263M in Example 2 was used as a template for site-directed mutagenesis at position 263. The site-directed mutagenesis was completed by whole plasmid PCR. Primer TylAT5-W263M-F: 5'-tggcgctgcgcagccgggcaatgctcacactggccggcaa-3' (SEQ ID NO. 9); TylAT5-W263M-R: 5'-tgcccggctgcgcagcgccacgatccgcgccgcgtcct-3' (SEQ ID NO. 10). After the completion of PCR, the template was digested with Dpnl, the PCR product was recovered by a column PCR product purification kit, and E. coli DH10B competent cells were transformed. The single colonies that grew were expanded in culture, and the plasmid pET28a-tylAT5 T335H / G337F / W263M was extracted using a plasmid extraction kit. After sequencing, E. coli BL21(DE3) competent cells were transformed, and the recombinant bacteria E. coli BL21(DE3) / pET28a-tylAT5 T335H / G337F / W263M were obtained. The three-point mutant amino acid sequence was SEQ ID NO. 3.
[0049] Example 5: Induced expression of acyltransferase wild type and mutants
[0050] The recombinant bacteria were cultured in 4 mL LB liquid medium (containing 50 mg / mL kanamycin) at 37°C, 220 rpm for 6 h, and then transferred into 1 L LB liquid medium (containing 50 mg / mL kanamycin) and cultured at 37°C, 220 rpm until the OD 600 was 0.4-0.6, and then induced overnight at 16°C after adding IPTG to a final concentration of 0.3 mM. The bacteria were centrifuged at room temperature at 5000 rpm for 7 min. The bacterial cells were resuspended in 100 mL of 50 mM Tris and 500 mM NaCl buffer, and then broken by ultrasonic treatment in an ice bath (10 s of breaking and 10 s of stopping, 10 min for one group) for two groups. The mixture was centrifuged at 4°C at 10000 rpm for 40 min, and the supernatant was collected. The target protein with a histidine tag was purified by nickel column affinity chromatography, and the supernatant was passed through the column twice. The impurities were eluted with 50 mL of 50 mM Tris, 500 mM NaCl and 20 mM imidazole buffer, and the target protein was eluted with 3 mL of 50 mM Tris, 500 mM NaCl and 300 mM imidazole buffer. The SDS-PAGE electrophoretogram of the wild-type recombinant bacteria is shown in Figure 2 , from left to right in the lanes are protein Marker, whole cell lysate, lysate precipitated protein, lysate supernatant protein, protein passing through the nickel column, 20 mM imidazole eluted impurities and target protein, wherein the target protein has a size of about 54 kDa.
[0051] Subsequently, the mixture was centrifuged at 4°C at 12000 rpm for 5 min, and further purified by fast protein liquid chromatography. A superdex200 10 / 300 column was used, with 10 mM Tris and 150 mM NaCl, pH 7.5 as the mobile phase, a flow rate of 0.5 mL / min, a sample volume of 1 mL, and the target protein was collected and placed in an ultrafiltration tube, which was concentrated to a protein concentration of 10 mg / mL by centrifugation at 4°C at 5000 rpm for 5 min.
[0052] Example 6: Determination of kinetic parameters of acyltransferase wild type and mutants
[0053] The determination method of enzyme activity: the total volume of the premix solution is 95 μL, containing 50 mM phosphate buffer (pH 7.6), 10% glycerol, 1 mM TCEP, 1 mM EDTA, 0.4 mU / μL α-KGDH, 0.4 mM NAD+, 0.4 mM TPP, 2 mM α-ketoglutarate, 2 μL of pure enzyme solution, 3 μL of substrate, and the change of absorbance at 340 nm with time is detected by spectrophotometer after mixing. In the process of measuring Km, the concentrations of protein and reactant NAD+ are fixed, and the reaction rates of 0, 6.25, 12.5, 25, 50, 100 μM substrate malonyl-CoA are measured to obtain Km. The kinetic parameters are shown in Table 1.
[0054] Table 1 Kinetic parameters
[0055]
[0056] The enzymatic reaction kinetic curves of acyltransferase TylAT5 wild type (TylAT5 WT) and mutants under different substrate concentrations are shown in Figure 3 The columnar chart of transacylation activity of acyltransferase TylAT5 wild type and mutants on malonyl-CoA is shown in Figure 4 From the above results, it can be seen that the activity of the two-point mutant provided in the application is increased to 1.86 times that of the wild type TylAT5, the activity of the three-point mutant is 2.4 times that of the wild type, and the activity of the four-point mutant is 2.6 times that of the wild type.
[0057] The preferred embodiments of the application are described in detail above. It should be understood that those of ordinary skill in the art can make many modifications and variations without creative labor based on the concept of the application. Therefore, any technical solution obtained by logical analysis, reasoning or limited experiment based on the prior art according to the concept of the application should be within the protection scope defined by the claims.
Claims
1. A tylosin polyketide synthase acyltransferase mutant, characterized in that, The tylosin polyketide synthase acyltransferase mutant is a first mutant, a second mutant, or a third mutant; the amino acid sequence of the first mutant is shown in SEQ ID NO.2, the amino acid sequence of the second mutant is shown in SEQ ID NO.3, and the amino acid sequence of the third mutant is shown in SEQ ID NO.
4.
2. The encoding gene of the tylosin polyketide synthase acyltransferase mutant according to claim 1.
3. A recombinant vector for constructing the encoding gene as described in claim 2.
4. A recombinant bacterium prepared by transformation of the recombinant vector according to claim 3.
5. The application of the tylosin polyketide synthase acyltransferase mutant of claim 1 in acyl transfer using malonyl-CoA as a substrate.
6. The application of the encoding gene of the tylosin polyketide synthase acyltransferase mutant according to claim 2 in acyl transfer using malonyl-CoA as a substrate.
7. The use of the recombinant vector according to claim 3 in acyl transfer using malonyl-CoA as a substrate.
8. The use of the recombinant bacteria of claim 4 in acyl transfer using malonyl-CoA as a substrate.