Spinosyn acyltransferase mutants and uses thereof

Abstract

The application discloses a spinosyn polyketide synthase acyltransferase mutant and application thereof, relates to the field of bioengineering, and comprises a first mutant, a second mutant or a third mutant; the amino acid sequences are shown in SEQ ID NO. 2, SEQ ID NO. 3 or SEQ ID NO. 4 respectively. The application discloses a coding gene, a recombination carrier and a recombination bacteria of the mutant and application of the mutant in production of spinosyn A with high purity and single component. The application discloses a soluble expression method of the acyltransferase domain SpnAT8 protein of the spinosyn polyketide synthase module eight, which comprises culturing bacteria, eluting the target protein and further purifying the target protein by using fast protein liquid chromatography. The four-point mutant provided by the application has the activity of recognizing malonyl-CoA restored to 19.5% compared with the wild type, and almost loses the activity of recognizing methylmalonyl-CoA.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bioengineering, and in particular to a mutant of spinosyn acyltransferase and its application. BACKGROUND

[0002] Spinosyn is a non-pollution high-efficiency insecticide extracted from the fermentation product of Saccharopolyspora spinosa, which mainly acts on the nicotinic acetylcholine receptor and the gamma-aminobutyric acid receptor, stimulates the nervous system of pests, causes non-functional muscle contraction, tremor, exhaustion and paralysis, and ultimately leads to the death of pests. Spinosyn can quickly and effectively kill Lepidoptera and other pests while having little impact on beneficial insects and other non-target organisms. The specific insecticidal effect, rapid degradation ability and biocompatibility of spinosyn make it a widely used biological pesticide. By 2019, more than 200 products of spinosyn have been registered in more than 100 countries.

[0003] The main components of spinosyn are spinosyn A and spinosyn D, and the insecticidal activity of spinosyn A on cotton aphids, red spider mites, tobacco aphid moths and other pests is better than that of spinosyn D. The difference between the two components is that the group connected at C6 is different, spinosyn A is connected with hydrogen, and spinosyn D is connected with methyl, and the ratio of the two components is about 85:155. The reason for this difference is the substrate selectivity of the acyltransferase domain in the eighth module of the polyketide synthase in the spinosyn biosynthetic gene cluster, which can recognize both malonyl-CoA and methylmalonyl-CoA. Because Saccharopolyspora spinosa has problems such as difficulty in cultivation, slow growth, and difficulty in genetic manipulation in the laboratory and industrial production, people mostly use metabolic engineering, directed breeding, heterologous host expression and semi-synthetic modification to produce and modify spinosyn. Ethyl spinosyn, a spinosyn derivative, is obtained by combining hydrogenation of the 5,6 double bond and 3'-O-ethylation of rhamnose. Butenyl spinosyn, another structural analogue, is a secondary metabolite of Saccharopolyspora pogona. These three spinosyn pesticides are mainly mixtures of two components, although the mixing ratio is different, but the structural difference of the components is the group at C6. The separation of spinosyn A and spinosyn D is difficult due to the particularity of the molecular structure, requires harsh equipment, has high cost, and is complex to operate. If the acyltransferase domain in the spinosyn biosynthetic gene cluster that determines the C6 group is modified to only recognize one substrate, malonyl-CoA, a higher-purity, single-component spinosyn A can be produced.

[0004] Acyltransferases act as "gatekeepers" in polyketide synthases, responsible for selecting and loading extension units. They are particularly important among the many structural domains, and various attempts have been made to modify polyketide synthases based on acyltransferases. Acyltransferase modification mainly includes substitution, site-directed mutagenesis, and cross-complementation. Substitution and cross-complementation affect the integrity of the overall polyketide synthase structure, ultimately leading to a significant reduction in polyketide synthase activity. In contrast, site-directed mutagenesis provides the most precise modification of acyltransferases, with the least impact on the overall polyketide synthase. With the development of sequence alignment methodologies, structural biology, and bioinformatics, site-directed mutagenesis technology for acyltransferases has been widely studied and applied. Current technology has not yet modified the substrate specificity of spinosad polyketide synthase acyltransferase to recognize only malonyl-CoA as a substrate, thereby producing higher purity and single-component spinosad A.

[0005] Therefore, those skilled in the art are dedicated to developing a substrate-specific modification of spinosad polyketide synthase acyltransferase and a mutant that recognizes only malonyl-CoA as a substrate, as well as its application methods. Summary of the Invention

[0006] In view of the above-mentioned deficiencies of the prior art, the technical problem to be solved by the present invention is to develop a substrate-specific modification of spinosad polyketide synthase acyltransferase and a mutant that recognizes only malonyl-CoA as a substrate and a method for its application.

[0007] To achieve the above objectives, the present invention provides a spinosad polyketide synthase acyltransferase mutant, including 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.

[0008] Furthermore, the first mutant of spinosad polyketide synthase acyltransferase is obtained by simultaneously mutating positions 303 and 305 of the amino acid sequence shown in SEQ ID NO.1; the second mutant is obtained by simultaneously mutating positions 205, 303, and 305 of the amino acid sequence shown in SEQ ID NO.1; and the third mutant is obtained by simultaneously mutating positions 205, 301, 303, and 305 of the amino acid sequence shown in SEQ ID NO.1.

[0009] Further, as shown in SEQ ID NO.1, tyrosine at position 303 is mutated to histidine and serine at position 305 is mutated to phenylalanine, resulting in the first mutant of spinosad polyketide synthase acyltransferase; then, based on the two-point mutation of the first mutant of spinosad polyketide synthase acyltransferase, glutamine at position 205 is mutated to valine, resulting in the second mutant of spinosad polyketide synthase acyltransferase; finally, based on the three-point mutation of the second mutant of spinosad polyketide synthase acyltransferase, isoleucine at position 301 is mutated to valine, resulting in the third mutant of spinosad polyketide synthase acyltransferase.

[0010] Furthermore, based on structure and sequence alignment, key amino acids in the active site were selected for site-directed mutagenesis. By modifying the acyltransferase domain SpnAT8 of spinosad polyketide synthase module 8, substrate specificity was successfully modified.

[0011] The present invention also provides a coding gene for a polyketide polyketide synthase acyltransferase mutant.

[0012] The present invention also provides a recombinant vector constructed from the encoding gene of a spinosad polyketide synthase acyltransferase mutant.

[0013] Furthermore, by determining the boundary of the spinosad polyketide synthase acyltransferase domain, the gene fragment range of the SpnAT8 acyltransferase domain of spinosad polyketide synthase module 8 was determined from the genome of *Saccharomyces cerevisiae*, and a recombinant expression vector containing the gene fragment was constructed.

[0014] Furthermore, based on sequence alignment, primers were designed to amplify the complete nucleic acid sequence encoding the acyltransferase domain and the linker fragment between the keto synthase and the acyltransferase, which was then ligated into the expression vector to obtain an expression vector containing the target gene, which can be used for the expression and purification of SpnAT8 protein.

[0015] The present invention also provides recombinant bacteria prepared by transformation of a recombinant vector constructed from the encoding gene of a spinosad polyketide synthase acyltransferase mutant.

[0016] This invention also provides a method for soluble expression of the acyltransferase domain SpnAT8 protein of spinosad polyketide synthase module 8, comprising the following steps:

[0017] Step 1: Incubate the recombinant bacteria in LB liquid medium at 37 ℃ on a shaker for 6 h; then transfer to LB liquid medium and incubate at 37 ℃ on a shaker until OD reaches 60%. 600 Add IPTG to a concentration of 0.4-0.6 and induce overnight at 16 °C; collect bacterial cells by centrifugation at room temperature.

[0018] Step 2: Resuspend the bacterial cells collected in Step 1 in buffer solution, sonicate on ice, centrifuge at 10,000 rpm for 40 min at 4 ℃, and collect the supernatant; purify the target protein with histidine tag by nickel column affinity chromatography, flow through the supernatant twice, elute the impurities with the first imidazole buffer, and elute the target protein with the second imidazole buffer to obtain the eluted solution;

[0019] Step 3: The eluted solution obtained in Step 2 was centrifuged at 4 °C and further purified by rapid protein liquid chromatography.

[0020] Further, in step 1, the LB liquid medium contains 50 mg / mL kanamycin, and the final concentration of added IPTG is 0.3 mM; in step 2, the buffer composition is 50 mM Tris and 500 mM NaCl, the first imidazole buffer composition is 50 mM Tris, 500 mM NaCl and 20 mM imidazole, and the second imidazole buffer composition is 50 mM Tris, 500 mM NaCl and 300 mM imidazole; in step 3, the centrifugation speed is 12000 rpm, and step 3 also includes: using a Superdex 200 10 / 300 column with 10 mM Tris and 150 mM NaCl, pH 7.5 as the mobile phase.

[0021] Furthermore, the optimal conditions for soluble expression of SpnAT8 were explored. A series of experimental procedures and conditions, including recombinant bacterial culture temperature, time, purification method, and reagents used, were determined. The protein containing the acyltransferase domain of spinosad polyketide synthase was expressed and purified in a soluble manner, yielding SpnAT8 protein, which exhibits good solubility, uniform state, and is a monomeric form of the spinosad polyketide synthase module eight acyltransferase domain. The high expression level and good state of SpnAT8 protein make it suitable for subsequent activity assays.

[0022] The present invention also provides an application of a spinosad polyketide synthase acyl acyl transferase mutant in the production of high-purity, single-component spinosad A, specifically by improving the activity of recognizing malonyl-CoA while reducing the activity of recognizing methylmalonyl-CoA.

[0023] The present invention also provides an application of the coding gene of a spinosad polyketide synthase acyl acyl transferase mutant in the production of high-purity, single-component spinosad A, specifically by improving the activity of recognizing malonyl-CoA while reducing the activity of recognizing methylmalonyl-CoA.

[0024] The present invention also provides an application of a recombinant vector containing a spinosad polyketide synthase acyl acyl transferase mutant in the production of high-purity, single-component spinosad A, specifically by improving the activity of recognizing malonyl-CoA while reducing the activity of recognizing methylmalonyl-CoA.

[0025] The present invention also provides an application of recombinant bacteria containing a spinosad polyketide synthase acyl acyl transferase mutant in the production of high-purity, single-component spinosad A, specifically by improving the activity of recognizing malonyl-CoA while reducing the activity of recognizing methylmalonyl-CoA.

[0026] In a preferred embodiment 1 of the present invention, the construction process of the recombinant strain of spinosad polyketide synthase acyl acyl transferase is described in detail.

[0027] In another preferred embodiment 2 of the present invention, the construction process of two-point mutation of spinosad polyketide synthase acyltransferase is described in detail.

[0028] In another preferred embodiment 3 of the present invention, the construction process of the three-point mutation of spinosad polyketide synthase acyltransferase is described in detail.

[0029] In another preferred embodiment 4 of the present invention, the construction process of the four-point mutation of spinosad polyketide synthase acyltransferase is described in detail.

[0030] In another preferred embodiment 5 of the present invention, the induced expression of spinosad polyketide synthase wild-type and mutant acyltransferase is described in detail.

[0031] In another preferred embodiment 6 of the present invention, the process of determining the kinetic parameters of spinosad polyketide synthase acyltransferase wild-type and mutant is described in detail.

[0032] The beneficial effects of this invention are as follows:

[0033] This invention modifies the substrate specificity of spinosad polyketide synthase acyltransferase, determining the gene fragment range of the SpnAT8 acyltransferase domain in module eight of spinosad polyketide synthase, and constructing a recombinant expression vector containing the gene fragment. This successfully modifies the substrate specificity, and the protein of the spinosad polyketide synthase acyltransferase domain is expressed and purified in a soluble manner. The activity recognizing malonyl-CoA is increased, while the activity recognizing methylmalonyl-CoA is decreased. This achieves the goal of recognizing only malonyl-CoA, enabling the production of higher purity, single-component spinosad A. The two-point mutant shows a decrease in malonyl-CoA recognition activity to 10% of the wild type, the three-point mutant recovers to 17.9% of the wild type, and the four-point mutant recovers to 19.5% of the wild type. Simultaneously, the two-point mutant showed a decrease in methylmalonyl-CoA recognition activity to 23.3% of the wild type, the three-point mutant activity to 6.2% of the wild type, and the four-point mutant activity to only 2.7% of the wild type. Compared to the wild type, the four-point mutant showed a recovery in malonyl-CoA recognition activity to 19.5%, almost completely losing its methylmalonyl-CoA recognition activity. An expression vector containing the target gene was obtained, which can be used for SpnAT8 protein expression and purification; a high-expression, well-formed SpnAT8 protein was obtained and used for activity assays. This invention provides a recombinant E. coli strain that efficiently expresses an acyltransferase mutant; compared to the wild-type acyltransferase, this mutant's efficiency in catalyzing methylmalonyl-CoA is only 2.74% of the wild type, at 0.0020 ± 0.0028 min. -1 μM -1 This provides a framework for the molecular modification of spinosad polyketide synthase acyltransferase.

[0034] The following will further explain the concept, specific structure, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0035] Figure 1 This is an electrophoresis image of the spnAT8 gene fragment from a preferred embodiment of the present invention;

[0036] Figure 2 This is an SDS-PAGE electrophoresis image of the SpnAT8 quadruple mutant recombinant bacteria from a preferred embodiment 5 of the present invention;

[0037] Figure 3 The kinetic curves of the enzymatic reaction of the acyltransferase SpnAT8 wild-type and mutant with malonyl-CoA in a preferred embodiment 6 of the present invention are shown.

[0038] Figure 4The kinetic curves of the enzymatic reaction of the acyltransferase SpnAT8 wild-type and mutant with methylmalonyl-CoA are shown in Example 6 of the present invention. Detailed Implementation

[0039] The following description, with reference to the accompanying drawings, illustrates several preferred embodiments of the present invention to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0040] Example 1: Construction of recombinant acyltransferase bacteria

[0041] Acyltransferases derived from *Saccharopolyspora spinosa* were selected. Using PCR technology with the *Saccharopolyspora spinosa* genome as a template, and primers spnAT8-F: 5'-ATCGTAATCCATATGggtaccaacgcacacctgat-3' (SEQ ID NO.5) and spnAT8-R: 5'-TGATTCGAATGAATTCActggaacgggtaggtcgg-3' (SEQ ID NO.6), the spnAT8 gene was amplified. NdeI and EcoRI restriction endonuclease sites were introduced at its 5' and 3' ends, respectively. The PCR reaction system (50 μL) consisted of: 25 μL 2×PhantaMax Buffer, 0.5 μL template DNA, 2 μL upstream primer, 2 μL downstream primer, 1 μL dNTPs, 2.5 μL DMSO, 1 μL PhantaMax Super-Fidelity DNA Polymerase, and 17 μL sterile water. The PCR reaction conditions were: 95 °C pre-denaturation for 3 min; 95 °C denaturation for 30 s, 60 °C annealing for 15 s, 72 °C extension for 1.5 min, for 28 cycles; 72 °C extension for 10 min. The PCR amplification product was verified by 1% agarose gel electrophoresis. When the band was approximately 1.4 kb and conformed to the size of SEQ ID NO.1, the PCR product was recovered using a DNA gel recovery kit. The recovered target gene fragment and the vector pET28a were double-digested with restriction endonucleases NdeI and EcoRI, respectively. After digestion, the PCR amplification fragment was recovered using a PCR product purification kit. The plasmid digestion products were subjected to 1% agarose gel electrophoresis and recovered using a DNA gel recovery kit. The spnAT8 gene fragment nucleic acid electrophoresis image is shown below. Figure 1As shown, the left lane contains the DNA marker, and the right lane contains the PCR amplification product of the spnAT8 gene. The recovered fragments and vector were ligated at a molar ratio of 1:3 and transformed into E. coli DH10B competent cells. Single colonies were picked and cultured to 4 mL. The plasmid pET28a-spnAT8 was extracted using a plasmid mini-extraction kit. After successful sequencing, it was transformed into E. coli BL21(DE3) competent cells to obtain recombinant E. coli BL21(DE3) / pET28a-spnAT8.

[0042] Example 2: Construction of two-point mutation of acyltransferase

[0043] The recombinant plasmid pET28a-spnAT8 from Example 1 was homology-aligned with the amino acid sequences of other acyltransferases in the spinosad synthesis gene cluster polyketide synthase, including SpnAT3 (recognizing methylmalonyl-CoA) and SpnAT1, SpnAT2, SpnAT4, SpnAT5, SpnAT6, SpnAT7, and SpnAT9 (recognizing malonyl-CoA). Corresponding mutation sites in SpnAT8 were identified, yielding the mutation sites Y303H, S305F, Q205V, and I301V. The conserved motif at the active site was mutated to obtain SpnAT8 Y303H / S305F. Site-directed mutagenesis was performed using whole-plasmid PCR. The template pET28a-spnAT8 was used as primers for whole plasmid PCR, with SpnAT8 Y303H / S305F-F:5'-cccggcgggtgggaattgatcatgccttccattcgccgcagatcgcg-3' (SEQ ID NO. 7) and SpnAT8 Y303H / S305F-R:5'-atcaattcccacccgccgggtctgcacgttttccgcgg-3' (SEQ ID NO. 8) as primers. The PCR reaction system (50 μL) consisted of: 25 μL 2×PhantaMax Buffer, 0.1 μL template DNA, 2 μL upstream primer, 2 μL downstream primer, 1 μL dNTPs, 2.5 μL DMSO, 1 μL PhantaMax Super-Fidelity DNA Polymerase, and 17.4 μL sterile water. The PCR reaction conditions were: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 60℃ annealing for 15 s, 72℃ extension for 7.5 min, for 25 cycles; and 72℃ extension for 10 min. The template was digested with DpnI, and the PCR product was recovered using a PCR product purification kit and transformed into *E. coli* DH10B competent cells. Single colonies were picked and cultured to a volume of 4 mL. The plasmid pET28a-spnAT8Y303H / S305F was extracted using a plasmid extraction kit. After successful sequencing, it was transformed into *E. coli* BL21(DE3) competent cells to obtain recombinant *E. coli* BL21(DE3) / pET28a-spnAT8Y303H / S305F. The two-point mutated amino acid sequence is SEQ ID NO.2.

[0044] Example 3: Construction of a three-point mutation of an acyltransferase

[0045] Using the recombinant plasmid pET28a-spnAT8Y303H / S305F from Example 2 as a template, site-directed mutagenesis was performed at position 205. Site-directed mutagenesis was completed using whole-plasmid PCR. Primers SpnAT8-Q205V-F: 5'-ccgcggtggtcggtcattccgtcggcgagatcgccgccgccgcc-3' (SEQ ID NO. 9); SpnAT8-Q205V-R: 5'-ggaatgaccgaccaccgcggcgggctccactccgcacg-3' (SEQ ID NO. 10), and the method was the same as in Example 2. After PCR, the template was digested with DpnI, and the PCR product was recovered using a PCR product purification kit and transformed into E. coli DH10B competent cells. Single colonies that grew were selected and expanded into larger cultures. The plasmid pET28a-spnAT8Y303H / S305F / Q205V was extracted using a plasmid extraction kit. After successful sequencing, it was transformed into E. coli BL21(DE3) competent cells to obtain the recombinant strain E. coli BL21(DE3) / pET28a-spnAT8Y303H / S305F / Q205V. The three-point mutant amino acid sequence is SEQ ID NO.3.

[0046] Example 4: Construction of a four-point mutation of an acyltransferase

[0047] Using the recombinant plasmid pET28a-spnAT8Y303H / S305F / Q205V from Example 2 as a template, site-directed mutagenesis was performed at position 301. Site-directed mutagenesis was completed using whole-plasmid PCR. Primers SpnAT8-I301V-F: 5'- tcccacccgccgggtctgcacgttttccgcggtcagtt-3' (SEQ ID NO. 11); SpnAT8-I301V-R: 5'- tgcagacccggcgggtgggagtcgatcatgccttccattcg-3' (SEQ ID NO. 12), and the PCR method was the same as in Example 2. After PCR, the template was digested with DpnI, and the PCR product was recovered using a PCR product purification kit and transformed into E. coli DH10B competent cells. Single colonies that grew were selected and expanded into larger cultures. The plasmid pET28a-spnAT8 Y303H / S305F / Q205V / I301V was extracted using a plasmid extraction kit. After successful sequencing, it was transformed into E. coli BL21(DE3) competent cells to obtain the recombinant strain E. coli BL21(DE3) / pET28a-spnAT8 Y303H / S305F / Q205V / I301V. The four-point mutant amino acid sequence is SEQ ID NO.4.

[0048] Example 5: Induced expression of wild-type and mutant acyltransferases

[0049] The recombinant bacteria were cultured in 4 mL LB liquid medium (containing 50 mg / mL kanamycin) at 37 ℃ and 220 rpm for 6 h, then transferred to 1 L LB liquid medium (containing 50 mg / mL kanamycin) and cultured at 37 ℃ and 220 rpm until OD reached. 600 The concentration was 0.6, and IPTG was added to a final concentration of 0.3 mM. Induction was performed overnight at 16 °C. The cells were harvested by centrifugation at 5000 rpm for 7 min at room temperature. The cells were resuspended in 100 mL of 50 mM Tris and 500 mM NaCl buffer, and sonicated in an ice bath (10 s for 10 s, 10 s for 10 min, one set for 10 min) for two sets. The cells were centrifuged at 10000 rpm for 40 min at 4 °C, and the supernatant was collected. The target protein with a histidine tag was purified by nickel column affinity chromatography. The supernatant was flow-throughd twice, and impurities were eluted with 50 mL of 50 mM Tris, 500 mM NaCl, and 20 mM imidazole buffer. The target protein was then eluted with 3 mL of 50 mM Tris, 500 mM NaCl, and 300 mM imidazole buffer. The SDS-PAGE electrophoresis image of the SpnAT8 tetramutant recombinant bacteria is shown below. Figure 2As shown, the lanes from left to right are: protein marker, whole cell lysate, protein precipitated by lysate, protein supernatant from lysate, protein flowing through a nickel column, eluted by 20 mM imidazole, target protein, and target protein after passing through a molecular sieve. The target protein is approximately 54 kDa in size.

[0050] Subsequently, after centrifugation at 12000 rpm for 5 min at 4 ℃, the protein was further purified by rapid protein liquid chromatography. A Superdex 200 10 / 300 column was used with a mobile phase of 10 mM Tris, 150 mM NaCl, and pH 7.5 at a flow rate of 0.5 mL / min and an injection volume of 1 mL. The target protein was collected in an ultrafiltration tube and concentrated to a protein concentration of 10 mg / mL by multiple centrifugations at 5000 rpm for 5 min at 4 ℃.

[0051] Example 6: Determination of kinetic parameters of wild-type and mutant acyltransferases

[0052] Enzyme activity assay: A 95 μL premix containing 50 mM Tris-HCl buffer (pH 7.5), 10% glycerol, 1 mM TCEP, 1 mM EDTA, 0.4 mU / μL α-KGDH, 0.4 mM NAD+, 0.4 mM TPP, and 2 mM α-ketoglutarate was added. 0.5 μL of purified enzyme solution and 2 μL of substrate were added, and the mixture was thoroughly mixed. The absorbance at 340 nm was measured over time using a spectrophotometer. During the Km measurement, the concentrations of protein and NAD+ were kept constant, and the reaction rates of 0, 6.25, 12.5, 25, 50, and 100 μM malonyl-CoA or methylmalonyl-CoA substrates were measured to obtain the Km. Kinetic parameters are shown in Table 1.

[0053] Table 1 Kinetic parameters of acyltransferase wild-type and mutant

[0054]

[0055] The enzymatic kinetic curves of the wild-type and mutant acyltransferase SpnAT8 to malonyl-CoA are shown in the figure below. Figure 3 As shown; the enzymatic kinetic curves of the acyltransferase SpnAT8 wild-type and mutant to methylmalonyl-CoA are as follows. Figure 4As shown in the results above, the two-point mutant provided in this application exhibits malonyl-CoA recognition activity reduced to 10% of the wild type, the three-point mutant recovers to 17.9% of the wild type, and the four-point mutant recovers to 19.5% of the wild type. Simultaneously, the two-point mutant exhibits methylmalonyl-CoA recognition activity reduced to 23.3% of the wild type, the three-point mutant activity reduced to 6.2% of the wild type, and the four-point mutant activity is only 2.7% of the wild type.

[0056] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A polyketide polyketide synthase acyltransferase mutant, characterized in that, The spinosad acyltransferase mutant is a second mutant or a third mutant; 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 spinosad polyketide synthase acyltransferase mutant as described in 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 as described in claim 3.

5. A method for soluble expression of the acyltransferase domain SpnAT8 protein of spinosad polyketide synthase module 8, characterized in that, The soluble expression method includes the following steps: Step 1: Incubate the recombinant bacteria described in claim 4 in LB liquid medium at 37 °C on a shaker for 6 h; then transfer to the LB liquid medium and incubate at 37 °C on a shaker until OD reaches 60%. 600 Add IPTG to a concentration of 0.6 and induce overnight at 16 °C; collect bacterial cells by centrifugation at room temperature. Step 2: Resuspend the bacterial cells collected in Step 1 in buffer solution, sonicate on ice, centrifuge at 4 ℃, and collect the supernatant; purify the target protein with histidine tag by nickel column affinity chromatography, flow through the supernatant twice, elute the impurities with the first imidazole buffer, and elute the target protein with the second imidazole buffer to obtain the eluted solution. Step 3: The eluted solution obtained in Step 2 was centrifuged at 4 °C and further purified by rapid protein liquid chromatography.

6. The soluble expression method as described in claim 5, characterized in that, In step 1, the LB liquid culture medium contains kanamycin at a concentration of 50 mg / mL, and the final concentration of the added IPTG is 0.3 mM. In step 2, the buffer solution consists of 50 mM Tris and 500 mM NaCl. The first imidazole buffer solution consists of 50 mM Tris, 500 mM NaCl, and 20 mM imidazole, and the second imidazole buffer solution consists of 50 mM Tris, 500 mM NaCl, and 300 mM imidazole. In step 3, the centrifugation speed is 12000 rpm. Step 3 also includes using a Superdex 200 10 / 300 column with a mobile phase of 10 mM Tris and 150 mM NaCl, and the pH of the mobile phase is 7.

5.

7. The application of the spinosad polyketide synthase acyltransferase mutant as described in claim 1 in the production of high-purity, single-component spinosad A.

8. The application of the coding gene of the spinosad polyketide synthase acyltransferase mutant as described in claim 2 in the production of high-purity, single-component spinosad A.

9. The application of a recombinant vector of the spinosad polyketide synthase acyltransferase mutant according to claim 3 in the production of high-purity, single-component spinosad A.

10. The application of a recombinant strain of the spinosad polyketide synthase acyltransferase mutant according to claim 4 in the production of high-purity, single-component spinosad A.

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