53 amino acid mutant of tylosin reductase and application of 53 amino acid mutant

Abstract

The invention relates to the technical field of protein engineering, in particular to a method for improving the yield and purity of tylosin A by constructing a mutant of tylosin reductase tylAKR and reducing the affinity of the mutant to a substrate. The Y53F mutant is obtained by carrying out directional mutation on amino acid at a key active site of the tylosin reductase, so that the activity of the tylosin reductase is inhibited, the reduction activity of the tylosin reductase to a substrate tylosin (component A) is reduced, the yield and purity of the tylosin (component A) are effectively improved, the dosage of veterinary antibiotics is reduced, and the production cost is reduced. The safety of animal husbandry and public health is guaranteed, and remarkable economic benefits and social benefits are achieved.

Description

Technical fields: This invention relates to the field of protein engineering technology, and in particular to a method for improving the yield and purity of tylosin A by constructing a mutant of tylosin reductase tylAKR to reduce its affinity for substrates. Background technology: Tylosin was discovered by Mac Fuire in 1960 from Streptomyces freundii (…). Streptomyces fradiae Tylosin, extracted from cultures, is a widely used veterinary antibiotic in veterinary drugs and feed additives. Tylosin fermentation products exist in several forms, including the main component tylosin (component A), desmycosin (component B), macrocin (component C), and relomycin (component D). Tylosin (component A) is the most important active component; therefore, the content of tylosin (component A) is the standard for measuring the quality of tylosin fermentation products. In actual production, the content of all four tylosin components must be greater than 95%, with tylosin (component A) reaching at least 80%.

[0001] The main tylosin-producing bacterium is *Streptomyces freundii*. Through years of strain selection and fermentation process optimization, the production level of tylosin through fermentation has been steadily improving. However, in the later stages of fermentation, the C20 aldehyde reduction reaction mediated by tylosin reductase leads to the irreversible conversion of some tylosin (fraction A) to ralomycin (fraction D), becoming a key factor limiting yield and purity. Although knocking out the tylosin reductase gene can terminate the reduction process of tylosin (fraction A) to ralomycin (fraction D), this also affects the normal growth of *Streptomyces freundii*, rendering this gene-knockout strain unproductive.

[0002] Tylosin reductase (tylAKR) can catalyze the reduction of tylosin to rylomycin. The wild-type tylosin reductase from Streptomyces fradiae is composed of 331 amino acid residues (including 996 bases), including two protein domains, with NADPH as a coenzyme, and belongs to the aldehyde-ketone reductase superfamily. In order to reduce the reductase activity of tylosin to rylomycin, Chinese patent application CN111925999A uses CRISPR / Cas9 technology to mutate the 642nd guanine of the tylosin reductase gene in Streptomyces fradiae ATCC19609 to adenine, forming a stop codon, so that the polypeptide chain terminates prematurely when the polypeptide chain extends to the 213th amino acid, forming an incomplete enzyme protein containing only one domain, which significantly reduces the reduction activity of tylosin, thereby reducing the content of rylomycin in the fermentation product. However, this technology does not completely inhibit the reduction of aldehyde-ketone reductase to the substrate, and the final product still contains rylomycin. Therefore, the present application further modifies the tylosin reductase containing the 213th amino acid obtained in the above-mentioned patent in order to further reduce the reduction activity of tylosin reductase.

[0003] The present application will inhibit the activity of tylosin reductase by directional mutation of the key active site amino acids of tylosin reductase, thereby reducing its enzyme activity and completely inhibiting the reduction of tylosin reductase to the substrate, so as to effectively improve the yield and purity of tylosin (component A), reduce the dosage of veterinary antibiotics, and protect the safety of animal husbandry and public health, with significant economic and social benefits. SUMMARY In order to solve the above technical problems, the present application reduces the enzyme activity of tylosin reductase by directional mutation of the key site amino acids of tylosin reductase, thereby completely inhibiting the reduction of tylosin reductase to the substrate, so that the fermentation end product of Streptomyces fradiae does not contain the by-product rylomycin.

[0004] The present application is achieved by the following technical route: One of the technical solutions provided by the present application is a tylosin reductase mutant, which is obtained by mutating the 53rd tyrosine to phenylalanine based on the tylosin reductase shown in SEQ ID NO. 1; the amino acid sequence of the mutant is shown in SEQ ID NO. 3; Further, at least one of the following mutations occurs in the above-mentioned mutant: the 55th tryptophan is mutated to alanine, or the 130th histidine is mutated to alanine; Further, the tylosin reductase mutant is any one of the following (1)-(4): (1) based on the tylosin reductase shown in SEQ ID NO. 1, the tyrosine at position 53 is mutated to phenylalanine (Y53F), denoted as tylAKR M2 ; (2) based on the tylosin reductase shown in SEQ ID NO. 1, the tyrosine at position 53 is mutated to phenylalanine, and the tryptophan at position 55 is mutated to alanine (Y53F-W55A), denoted as tylAKR M8 ; (3) based on the tylosin reductase shown in SEQ ID NO. 1, the tyrosine at position 53 is mutated to phenylalanine, and the histidine at position 130 is mutated to alanine (Y53F-H130A), denoted as tylAKR M9 ; (4) based on the tylosin reductase shown in SEQ ID NO. 1, the tyrosine at position 53 is mutated to phenylalanine, the tryptophan at position 55 is mutated to alanine, and the histidine at position 130 is mutated to alanine (Y53F-W55A-H130A), denoted as tylAKR M13 .

[0005] The second technical solution provided by the present application is a coding gene of the tylosin reductase mutant of the first technical solution.

[0006] The third technical solution provided by the present application is a recombinant vector or a recombinant strain comprising the coding gene of the second technical solution. Further, the expression plasmid used by the recombinant vector is pET-28a. Further, the host used by the recombinant strain is Escherichia coli or Streptomyces fradiae.

[0007] The fourth technical solution provided by the present application is the application of the recombinant vector or the recombinant strain of the third technical solution in producing the tylosin reductase mutant of the first technical solution.

[0008] The fifth technical solution provided by the present application is the application of the tylosin reductase mutant of the first technical solution in reducing the conversion capacity of a production strain to tylosin (component A) to relomycin (component D). Further, the application is achieved by mutating the tylosin reductase coding gene in the production strain to the coding gene of the tylosin reductase mutant of the first technical solution. Further, the production strain is Streptomyces fradiae; preferably CICC 11034.

[0009] The sixth technical solution provided by the application is a Streptomyces fradiae genetically engineered bacterium, wherein the original tylosin reductase coding gene on the genome of the Streptomyces fradiae is mutated into the coding gene of the tylosin reductase mutant in the first technical solution. Further, the Streptomyces fradiae starting strain is numbered CICC 11034.

[0010] The seventh technical solution provided by the application is an application of the Streptomyces fradiae genetically engineered bacterium in the sixth technical solution in production of tylosin. Further, the tylosin produced by the Streptomyces fradiae genetically engineered bacterium does not contain a by-product, i.e., rylomycin.

[0011] Beneficial effects: The tylosin reductase mutant obtained by the Y53F mutation of the tylosin reductase shown in SEQ ID NO. 1 and further combined mutations at W55A and H130A sites has a significantly reduced reductase activity. When the mutant is applied to production of tylosin, the Streptomyces fradiae fermentation end product does not contain a by-product, i.e., rylomycin, thereby effectively improving the yield and purity of tylosin A, which is conducive to reducing the dosage of veterinary antibiotics, ensuring the safety of animal husbandry and public health, and having significant economic and social benefits. BRIEF DESCRIPTION OF DRAWINGS Figure 1 The growth curves of different strains. DETAILED DESCRIPTION The technical content of the application will be further described below in combination with examples, but the application is not limited to the examples, and the protection scope of the application cannot be limited by the examples.

[0012] In the application, the following definitions are adopted: 1. Nomenclature of amino acid and DNA nucleic acid sequences The recognized IUPAC nomenclature of amino acid residues is used in the form of single letter / three letter code. The recognized IUPAC nomenclature of DNA nucleic acid sequences is used.

[0013] 2. Identification of tylosin reductase mutant The mutated amino acid in tylosin reductase mutants is represented by "original amino acid + position + substituted amino acid". For example, Y53F indicates that the amino acid at position 53 is mutated from tylosin reductase tyrosine to phenylalanine, and the position number corresponds to the amino acid sequence number of tylosin reductase in SEQ ID NO: 1. tylAKR represents the original tylosin reductase (which is a tylosin reductase containing only 213 amino acids obtained by mutating guanine at position 642 to adenine, forming a stop codon, thus prematurely terminating polypeptide chain translation at amino acid position 213, named original tylAKR). tylakr This represents the gene encoding the primitive tylosin reductase.

[0014] The original tylosin reductase and the names and sequences of its mutants are shown in Table 1.

[0015] Table 1: Mutant Sequence Comparison Table

[0016] The amino acid sequence of the original tylAKR is shown in SEQ ID NO.1: MEYTQLGRIGLKVSRLVLGTMNFGPTTDEAESHAIMDAALDAGINFFDTANVYGWGENKGRTEEILGSWFAQGGDRRDKVVLATKVYGNMGLDGPAWPNHDKLSAL NIRRSVDASLKRLGTDHIDLYQFHHVDRDTPWDEIWQAMDVLVRQGKILYVGSSNFAGWNIAQANETAARHGRLGLVSEQCLYNLCERRAEMEVVPAAREYGLGVIA The Y53F mutation-inducing tylAKR M2 The amino acid sequence is shown in SEQ ID NO.3: MEYTQLGRIGLKVSRLVLGTMNFGPTTDEAESHAIMDAALDAGINFFDTANVFGWGENKGRTEEILGSWFAQGGDRRDKVVLATKVYGNMGLDGPAWPNHDKLSAL NIRRSVDASLKRLGTDHIDLYQFHHVDRDTPWDEIWQAMDVLVRQGKILYVGSSNFAGWNIAQANETAARHGRLGLVSEQCLYNLCERRAEMEVVPAAREYGLGVIA Table 2. Some primers and sequences involved in the embodiments of the present invention.

[0017] The application will be further explained by specific examples below.

[0018] Construction and expression of tylosin reductase and mutant genes 1. tylAKR and tylAKR M2 , tylAKR M8 , tylAKR M9 , tylAKR M13 Obtaining of coding genes Original tylAKR, and four mutant tylAKR M2 , tylAKR M8 , tylAKR M9 , tylAKR M13 Coding genes of original tylAKR, and four mutant tylAKR M2 , tylAKR M8 , tylAKR M9 , tylAKR M13 , tylAKR were obtained by artificial synthesis method (synthesized by Jinweizhi Biotechnology Co., Ltd.) according to the sequences shown in SEQ ID NO. 2, 4, 6, 8, 10, respectively, and PCR amplified by primers 213AA-F and 213AA-R.

[0019] 2. Construction of prokaryotic expression vector The prokaryotic expression vector used in this study is pET-28a. The vector is linearized by PCR, and the primers used are 28A-F and 28A-R. The linearized vector and the target gene tylakr , tylakr M2 , tylakr M8 , tylakr M9 , tylakr M13 are connected using Clon Express® II One Step Cloning Kit C115 enzyme, respectively. The connection liquid is transferred into DH5a competent cells, spread on a plate containing 50 μg / ml kanamycin, incubated at 37°C overnight, and positive clones are detected and screened for enzyme digestion and sequencing verification. Recombinant vectors pET-28a-tylAKR, pET-28a-tylAKR M2 , pET-28a-tylAKR M8 , pET-28a-tylAKR M9 , pET-28a-tylAKR M13 are obtained.

[0020] The above recombinant vectors are respectively transformed into E. coliThe positive clones were screened and sequenced to obtain the recombinant expression strain.

[0021] 3. Induction expression and purification of recombinant protein The above recombinant strains were inoculated into LB medium containing 50 μg / mL kanamycin at a ratio of 1:100, and cultured at 37°C and 150 r / min until the OD 600 When the OD reached 0.6, 0.2 mmol / L IPTG was added, and the culture was induced at 18°C for 20 h. The bacterial solution was centrifuged at 6000 r / min for 5 min, and the supernatant was discarded until the bacterial cells were collected. The cells were resuspended in buffer A (50 mM Tris, pH 7.5, 300 mM NaCl), and the cells were completely broken by a cell disrupter. The supernatant was collected by high-speed centrifugation.

[0022] Purification was performed by Ni column affinity chromatography. After equilibration of the Ni column with buffer A, the supernatant was added. Non-specific binding proteins were removed using buffer A and buffer B at a volume ratio of 9:1. The target protein was eluted using buffer B (50 mM Tris, pH 7.5, 300 mM NaCl, 500 mM imidazole). The eluate was collected as the purified target protein. Five purified target proteins were obtained in this step.

[0023] 4. Determination of enzyme activity by high performance liquid chromatography 1 mL of enzyme reaction system: enzyme (5 μM), NADPH (1.5 mM), tylosin (2.5 mM), supplemented with 100 mmol / L phosphate buffer (pH=7) to 1 mL, 30°C reaction for 10 min, without enzyme reaction as blank control, 1 min catalytic reduction of 1 μmol Tylosin enzyme amount was defined as 1 enzyme activity unit (U), the calculation formula was as follows:

[0024] ΔC: change of substrate concentration in the reaction system (μM); t: reaction time (s);

[0025] Ce: enzyme concentration (mg / mL) V: enzyme volume (mL) The reaction solution was filtered and prepared for loading. The chromatographic column was a Shimadzu C18 liquid chromatography column. The consumed tylosin (tylosin A component) in the reaction system was determined. The mobile phase was sodium perchlorate-acetonitrile (60:40), and the flow rate was 1.00 ml / min. The injection volume was 20 µL, and the ultraviolet detection wavelength was 280 nm. The high-performance liquid chromatography tylosin determination standard curve was fitted, and the activity of each protease was calculated.

[0026] The results are shown in Table 3 below. After mutation, the tylosin reductase still has a small amount of activity, but the activity is significantly reduced.

[0027] Table 3

[0028] Example 2 Obtaining Streptomyces fradiae SF106 1. Obtaining homologous arms The genomic DNA of Streptomyces fradiae ATCC 19609 (available from China General Microbiological Culture Collection Center, CICC11034) was used as the template. The primers 642-U-F / 642-U-R and 642-D-F / 642-D-R were used to amplify the sequences of 1 kb upstream and downstream of the 642nd base of the wild-type tylosin reductase encoding gene as the upstream and downstream homologous arms.

[0029] The plasmid used was pCRISPomyces-2, and the enzyme cutting site Xba I. The homologous arms were connected to the plasmid using the Clon Express® II One Step Cloning Kit C115 enzyme, and the LB plate was coated and incubated at 37 ℃ overnight until single colonies grew.

[0030] A single colony was picked and expanded in LB medium, and enzyme cutting verification was performed to obtain a 2 kb upstream and downstream homologous arm band. The vector successfully connected the homologous arms to pCRISPomyces-2 was obtained and named pC-642-HR.

[0031] 2. Connection of sgRNA The sgRNA of the target gene was designed using snapgene software, and was 5'-GCGACTAGGCGATGGCCCCG-3' and 5'-CCCGCCGTGGAGCGGCGATC-3', respectively. The sgRNA was inserted into the Bbs I site of pC-642-HR using the Golden Gate assembly method. After blue-white spot screening, PCR verification was performed using the detection primers JC-F / JC-R, and the target band was 3362 bp, indicating that the sgRNA was successfully inserted into the vector. The successfully constructed mutant vector was named pC-642-HR-sgRNA.

[0032] 3. Obtaining Streptomyces fradiae SF106 The pC-642-HR-sgRNA plasmid was transformed into ET12567 (pUZ8002) E. coli competent cells, and a single colony was picked and conjugated with Streptomyces fradiae ATCC 19609: Donor strain: ET12567 E. coli competent cells containing the pC-642-HR-sgRNA vector were inoculated in LB medium at 37°C for 3-4 h. 5 mL of the bacterial solution was centrifuged to remove the supernatant. 5 mL of LB was added, vortexed, and centrifuged to remove the supernatant, and the operation was repeated twice. Finally, 500 μL of LB was added and mixed well with a pipette.

[0033] Recipient strain: Streptomyces fradiae ATCC 19609 was plated on WL-50 solid medium. An appropriate amount of spores was scraped and inoculated in TSB medium, and the medium was incubated at 28°C until the mycelium was uniformly dispersed in the TSB medium. 5 mL of the bacterial solution was centrifuged to remove the supernatant. An equal volume of 10% glycerol was added, centrifuged to remove the supernatant, and the mycelium was washed. An equal volume of 2×YT medium was added, centrifuged to remove the supernatant, and the washing operation was repeated twice. Finally, 500 μL of 2×YT was added and resuspended, and the mixture was mixed well.

[0034] The 500 μL Streptomyces fradiae ATCC 19609 mycelium was mixed with 500 μL of ET12567 E. coli competent cells containing the pC-642-HR-sgRNA vector, and the mixture was centrifuged to remove the supernatant. 100 μL of the mixed bacterial solution was plated on WL-50 plates without antibiotics, and after 18 h of incubation in a 28°C incubator, antibiotic overlay was performed. The conjugants were placed in a 28°C incubator and incubated until the conjugants grew. After three passages, a single colony that produced spores was picked, and positive conjugants were selected. The tylosin reductase gene at position 642 in the strain was mutated from G to A to form a stop codon (i.e., the tylosin reductase gene at position 642 in Streptomyces fradiae ATCC 19609 was mutated from guanine to adenine to form a stop codon, so that the polypeptide chain translation was terminated when the peptide chain extended to the 213th amino acid). The mutant strain was named Streptomyces fradiae SF106. The tylosin reductase expression product of the strain is shown in SEQ ID NO. 1, and only contains 213 amino acids.

[0035] Example 3. Site-directed mutation of the tylosin reductase gene in Streptomyces fradiae SF106 (Y53F) 1. Obtaining homologous arms The Streptomyces fradiae SF106 genomic DNA was used as a template, and Y53-U-F / Y53-U-R and Y53-D-F / Y53-D-R were used as primers to amplifytylakr The 1kb sequences upstream and downstream of positions 157-159 of the coding gene were used as upstream and downstream homologous arms. The plasmid used in this study was pCRISPomyces-2, with the following restriction enzyme sites: Xba Ⅰ. Use the Clon Express® II One Step Cloning Kit C115 enzyme to ligate the homologous arm to the plasmid, spread it on LB agar plates, and incubate overnight at 37 °C until a single colony grows.

[0036] Single colonies were picked and cultured on LB medium for expansion. Enzyme digestion was performed for verification, yielding 2kb upstream and downstream homologous arm bands. If the verification was successful, the vector with the homologous arms successfully ligated to pCRISPomyces-2 was obtained and named pC-Y53F-HR.

[0037]

[0038] 2. Ligation of sgRNA Designed using snapgene software tylakr The target gene's sgRNAs were 5'-CTCGCCCCAGCCGTAGACGT-3' and 5'-TGCGGCCCTTGTTCTCGCCC-3', respectively. The sgRNAs were inserted into pC-Y53F-HR using the Golden Gate assembly method. Bbs At site I. After blue-white screening, PCR verification was performed using detection primers JC-F / JC-R. The target band was 3362 bp, indicating that the sgRNA was successfully inserted into the vector. The successfully constructed mutant vector was named pC-Y53F-HR-sgRNA.

[0039]

[0040] 3. Obtaining the SF Y53F mutant The pC-Y53F-HR-sgRNA plasmid was transformed into ET12567 (pUZ8002) Escherichia coli competent cells, and single colonies were picked for conjugation transfer with Streptomyces f. flexneri SF106. Donor strain: ET12567 competent E. coli cells containing the pC-Y53F-HR-sgRNA vector were inoculated into LB medium and incubated at 37 °C for 3–4 h with shaking. 5 mL of the bacterial culture was collected, centrifuged, and the supernatant was discarded. 5 mL of LB medium was added, vortexed, and the supernatant was discarded; this process was repeated twice. Finally, 500 μL of LB medium was added, and the mixture was thoroughly mixed by pipetting.

[0041] Receptor strain: Streptomyces fradiae SF106 was coated on WL-50 solid plate. A suitable amount of spores was inoculated in TSB medium, and the mycelium was uniformly dispersed in the TSB medium at 28°C. 5 mL of bacterial solution was centrifuged to remove the supernatant; 10% glycerol was added in an equal volume, and the supernatant was removed by centrifugation to wash the mycelium; 2xYT medium was added in an equal volume, and the supernatant was removed by centrifugation, and the washing was repeated twice; finally, 500 μL of 2xYT was added for resuspension, and the mixture was thoroughly mixed.

[0042] 500 μL of Streptomyces fradiae SF106 mycelium was mixed with 500 μL of ET12567 E. coli competent cells containing pC-Y53F-HR-sgRNA vector, and the supernatant was removed by centrifugation. 100 μL of mixed bacterial solution was coated on WL-50 plate without antibiotics, and after 18 h culture in 28°C incubator, antibiotic overlay was performed, and the culture was placed in 28°C incubator for inversion culture until the combination of zygotes was grown. After three generations of zygotes, single colonies capable of producing spores were selected, and positive zygotes were screened to obtain the strain tylakr The TAC at positions 157-159 of the coding gene was mutated to TTC to obtain the mutant strain SFY53F, and the expression product of the tylosin reductase of the strain is shown in SEQ ID NO. 3.

[0043] Example 4 Site-directed mutation (W55A) of tylosin reductase gene in Streptomyces fradiae SF106 1. Obtaining of homologous arms The genomic DNA of Streptomyces fradiae SF106 was used as a template, and the primers W55-U-F / W55-U-R and W55-D-F / W55-D-R were used for amplification tylakr The sequences of 1 kb upstream and downstream of the bases at positions 163-165 of the coding gene were used as the upstream and downstream homologous arms. The plasmid used in this study was pCRISPomyces-2, and the enzyme cutting site Xba Ⅰ, the homologous arms were connected to the plasmid using Clon Express® II One Step Cloning Kit C115 enzyme, and coated on LB plate, and incubated at 37°C overnight until single colonies were grown.

[0044] A single colony was picked and expanded in LB medium, and enzyme digestion was performed to obtain a 2 kb upstream and downstream homologous arm band, and the vector successfully connected with the homologous arms was obtained and named pC-W55A-HR.

[0045]

[0046] 2. Connection of sgRNA The sgRNA was designed using snapgene software tylakrsgRNAs of the target gene were 5'-ACCGCCAACGTCTACGGCGC-3' and 5'-CTCGCCTGCGCCGTAGACGT-3', respectively. The sgRNAs were inserted into the pC-W55A-HR at the Ⅰ site by the Golden Gate assembly method. Bbs After blue-white spot screening, PCR verification was performed using detection primers JC-F / JC-R, and the target band was 3362 bp, indicating that the sgRNA was successfully inserted into the vector. The successfully constructed mutant vector was named pC-W55A-HR-sgRNA.

[0047]

[0048] 3. Obtaining of W55A mutant The pC-W55A-HR-sgRNA plasmid was transformed into ET12567 (pUZ8002) E. coli competent cells, and a single colony was selected for conjugation transfer with Streptomyces fradiae SF106: Donor strain: The ET12567 E. coli competent cells containing the pC-W55A-HR-sgRNA vector were inoculated in LB medium at 37 °C for 3-4 h. 5 mL of bacterial solution was centrifuged to remove the supernatant. 5 mL of LB was added and vortexed, and the supernatant was removed by centrifugation twice. Finally, 500 μL of LB was added and mixed well by blowing with a pipette.

[0049] Recipient strain: Streptomyces fradiae SF106 was plated on WL-50 solid plates. An appropriate amount of spores was inoculated in TSB medium, and the culture was incubated at 28 °C until the mycelium was evenly dispersed in the TSB medium. 5 mL of bacterial solution was centrifuged to remove the supernatant; an equal volume of 10% glycerol was added, and the mycelium was washed by centrifugation; an equal volume of 2×YT medium was added, and the supernatant was removed by centrifugation, and the washing was repeated twice; finally, 500 μL of 2×YT was added and resuspended, and mixed well.

[0050] 500 μL of Streptomyces fradiae SF106 mycelium was mixed with 500 μL of ET12567 E. coli competent cells containing the pC-W55A-HR-sgRNA vector, and the supernatant was removed by centrifugation. 100 μL of the mixed bacterial solution was applied to an antibiotic-free WL-50 plate, and after 18 h of incubation in a 28 °C incubator, antibiotic overlay was performed, and the conjugants were incubated in a 28 °C incubator until spores were produced. After three passages of the conjugants, a single colony that could produce spores was selected, and positive conjugants were screened to obtain a strain tylakrThe TGG at positions 163-165 of the coding gene is mutated to GCA to obtain mutant strain SFW55A, and the expression product of tylosin reductase of the strain is a W55A mutant based on SEQ ID NO. 1.

[0051] Example 5 Site-directed mutation (H130A) of tylosin reductase gene in Streptomyces fradiae SF106 1. Obtaining of homologous arms The genomic DNA of Streptomyces fradiae SF106 was used as a template, and H130-U-F / H130-U-R and H130-D-F / H130-D-R were used as primers to amplify tylakr The sequence of 1 kb upstream and downstream of the base at positions 388-390 of the coding gene was used as the upstream and downstream homologous arms. The plasmid used in this study was pCRISPomyces-2, and the enzyme cutting site Xba I, the homologous arms were ligated with the plasmid using Clon Express® II One Step Cloning Kit C115 enzyme, and plated on LB plates and incubated at 37°C overnight to grow single colonies.

[0052] A single colony was picked and expanded in LB medium, and enzyme digestion verification was performed, and a 2 kb upstream and downstream homologous arm band was obtained. Verification was correct, and the homologous arm and pCRISPomyces-2 ligated successfully were obtained, and named pC-H130A-HR.

[0053]

[0054] 2. Connection of sgRNA The sgRNA of the target gene was designed using snapgene software, which was 5'-CTCGCCCCAGCCGTAGACGT-3' and 5'-TGCGGCCCTTGTTCTCGCCC-3', respectively. The sgRNA was inserted into the tylakr I site of pC-Y53F-HR using the Golden Gate assembly method. After blue-white spot screening, PCR verification was performed using detection primers JC-F / JC-R, and the target band was 3362 bp, indicating that the sgRNA was successfully inserted into the vector, and the successfully constructed mutant vector was named pC-H130A-HR-sgRNA. Bbs

[0055]

[0056] 3. Obtaining of H130A mutant ​The pC-H130A-HR-sgRNA plasmid is transformed into ET12567 (pUZ8002) E. coli competent cells, and a single colony is selected for conjugation transfer with Streptomyces fradiae SF106: The donor strain: ET12567 E. coli competent cells containing the pC-H130A-HR-sgRNA vector are inoculated in LB medium at 37°C for 3-4 h. 5 mL of the bacterial solution is centrifuged to remove the supernatant. 5 mL of LB is added, vortexed, and centrifuged to remove the supernatant, and the process is repeated twice. Finally, 500 μL of LB is added and mixed well with a pipette.

[0057] The recipient strain: Streptomyces fradiae SF106 is plated on WL-50 solid plates. An appropriate amount of spores is inoculated in TSB medium, and the culture is incubated at 28°C until the mycelium is uniformly dispersed in the TSB medium. 5 mL of the bacterial solution is centrifuged to remove the supernatant; an equal volume of 10% glycerol is added, and the mycelium is washed by centrifugation to remove the supernatant; an equal volume of 2×YT medium is added, and the mycelium is washed by centrifugation to remove the supernatant, and the process is repeated twice; finally, 500 μL of 2×YT is added and resuspended, and the mixture is mixed well.

[0058] The 500 μL Streptomyces fradiae SF106 mycelium is mixed with 500 μL ET12567 E. coli competent cells containing the pC-H130A-HR-sgRNA vector, and the mixture is centrifuged to remove the supernatant. 100 μL of the mixed bacterial solution is applied to an antibiotic-free WL-50 plate, and after 18 h of incubation in a 28°C incubator, antibiotic coverage is performed, and the plate is incubated in a 28°C incubator until the conjugants grow. After three passages of the conjugants, a single spore-producing colony is selected, and the positive conjugants are screened to obtain a strain tylakr The CAC at positions 388-390 of the coding gene is mutated to GCA to obtain the mutant strain SFH130A, and the tylosin reductase expression product of this strain is mutated at H130A based on SEQ ID NO. 1.

[0059] The construction methods of the remaining double / multiple mutations are basically the same as those of the single mutation, and only need to be superimposed on the basis of the single mutation.

[0060] Example 6: Growth curve determination and tylosin production capacity verification 1. Growth curve determination The growth curves of mutant strains SFY53F, SF106, and knock-out strain ΔtytlAKR (a strain obtained by knocking out the tylosin reductase gene in SF106) are determined as follows: Into a 250 mL flask containing TSB liquid medium (yeast powder 5 g / L, tryptone 15 g / L, soybean peptone 5 g / L, sodium chloride 5 g / L), inoculate with an appropriate amount of spores of the test strain, and cultivate at 200 r / min, 28°C for 48 h to obtain a seed solution. Inoculate the seed solution into TSB liquid medium again at a 5% (v / v) inoculation amount, and cultivate at 150 r / min, 28°C for 10 d, taking a sample every 12 h, collecting mycelium, and determining the dry weight of the mycelium. Plot a growth curve with time as the abscissa and the dry weight of the mycelium as the ordinate.

[0061] The results are shown in Table 1. Figure 1 The mutant strain (SFY53) grows normally, indicating that the mutation does not adversely affect the growth of the bacterial body and can be used for industrial research and tylosin production. The knockout strain ΔtytIAKR cannot grow normally, indicating that tylosin cannot be converted to rylomycin by directly knocking out the tylosin reductase gene.

[0062] 2. Verification of the tylosin production capacity of the mutant strain Ferment the strains SF106 and the mutant strain SFY53F, specifically as follows: Scrape 4×4 cm 2 Inoculate spores into fermentation medium, and cultivate at 200 r / min, 28°C for 168 h (fermentation medium: fish meal 6.6 g / L, corn syrup 30 g / L, CaCO32.2 g / L, KCl 1.1 g / L, soybean oil 41.0 g / L, betaine 0.72 g / L, (NH4)2HPO40.44 g / L, NiSO4•6H2O 0.0044 g / L, CoCl2•6H2O 0.0033 g / L, MgSO4•7H2O 0.1 g / L) After fermentation in the fermentation medium for 168 h, filter the fermentation solution after acidification with an aluminum sulfate solution, and detect tylosin (component A) and rylomycin (component D) in the fermentation solution by HPLC. The comparison of the detection results of the tylosin and rylomycin contents in the fermentation solutions is shown in the following table.

[0063]

[0064] Note: The above "%" is calculated according to the relative peak area of each component in the total components in the high-performance liquid chromatogram.

[0065] The results show that, compared with the strain SF106, the percentage of tylosin in the total components of the mutant strain SFY53F is increased from 91.20% to 94.46%, and the percentage of rylomycin in the total components is decreased from 4.19% to 0.00%. It can be seen that the tylosin reductase mutant provided by the present application has completely lost the reductase activity, and the product does not contain rylomycin, thereby effectively improving the yield and purity of tylosin (component A).

[0066] Although the present application has been disclosed in the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various changes, modifications, replacements and variations in form and details to these embodiments without departing from the spirit and principles of the present application, and the scope of the present application is defined by the claims and their equivalents.

Claims

1. A tylosin reductase mutant, characterized in that, The mutant was obtained by mutating tylosin reductase at position 53 to phenylalanine; the amino acid sequence of the mutant is shown in SEQ ID NO.

3.

2. The tylosin reductase mutant as described in claim 1, characterized in that, The tylosin reductase mutant also contains at least one of the following mutation sites: tryptophan at position 55 is mutated to alanine, or histidine at position 130 is mutated to alanine.

3. The tylosin reductase mutant as described in claim 2, characterized in that, The tylosin reductase mutant is any one of the mutants described in (1)-(4) below: (1) Based on the tylosin reductase shown in SEQ ID NO.1, the tyrosine at position 53 is mutated to phenylalanine (Y53F), denoted as tylAKR. M2 ; (2) Based on the tylosin reductase shown in SEQ ID NO.1, tyrosine at position 53 is mutated to phenylalanine, and tryptophan at position 55 is mutated to alanine (Y53F-W55A), denoted as tylAKR. M8 ; (3) Based on the tylosin reductase shown in SEQ ID NO.1, the tyrosine at position 53 is mutated to phenylalanine, and the histidine at position 130 is mutated to alanine (Y53F-H130A), denoted as tylAKR. M9 ; (4) Based on the tylosin reductase shown in SEQ ID NO.1, tyrosine at position 53 is mutated to phenylalanine, tryptophan at position 55 is mutated to alanine, and histidine at position 130 is mutated to alanine (Y53F-W55A-H130A), denoted as tylAKR. M13 .

4. The encoding gene of the tylosin reductase mutant according to any one of claims 1-3.

5. A recombinant vector or recombinant strain comprising the encoding gene of claim 4.

6. The use of the recombinant vector or recombinant strain according to claim 5 in the production of the tylosin reductase mutant according to any one of claims 1-3.

7. The application of the tylosin reductase mutant according to any one of claims 1-3 in reducing the conversion ability of the producing strain of tylosin to raloxim, characterized in that, The application is achieved by mutating the tylosin reductase encoding gene in the production strain to the encoding gene of the tylosin reductase mutant according to any one of claims 1-3.

8. A genetically engineered strain of *Streptomyces freundii*, characterized in that, The engineered bacteria are obtained by mutating the original tylosin reductase encoding gene in the genome of *Streptomyces freundii* to the encoding gene of the tylosin reductase mutant as described in any one of claims 1-3.

9. The engineered bacteria as described in claim 8, characterized in that, The Streptomyces freundii strain was numbered CICC11034.

10. The use of the engineered bacteria according to claim 8 in the production of tylosin.

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