Method for increasing lincomycin yield by knocking out streptomyces lincomycin pAgene0296 gene

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a method for increasing the yield of lincomycin by knocking out a streptomyces lincomycin pAgene0296 gene. The method comprises the following steps: knocking out a coding gene pAgene0296 of TetR / AcrR family repressor protein in streptomyces lincomycin, and fermenting the obtained mutant strain to produce lincomycin. Experimental results show that after the pAgene0296 gene of the streptomyces lincomycin is knocked out, the yield of the lincomycin is remarkably increased. The invention further provides a construction method of the pAgene0296 gene deletion type streptomyces lincoli mutant strain, according to the method, the coding gene of the streptomyces lincoli is directionally modified through a gene engineering technology, so that the pAgene0296 gene is knocked out, and the obtained pAgene0296 gene deletion type streptomyces lincoli mutant strain can be used for efficiently producing lincomycin or a product in the synthesis process of the lincomycin.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for increasing the yield of lincomycin by knocking out a pA_gene0296 gene of Streptomyces lincomycin. Background Art

[0002] Lincomycin is a lincosamide antibiotic that acts on the central site of the peptidyl transferase of bacterial ribosomes, interacts with the A site and P site of the 50S ribosomal subunit, hinders the positioning of two tRNA molecules, and inhibits bacterial growth by directly inhibiting the formation of peptide bonds and the extension of peptide chains during protein synthesis in bacteria. It is a broad-spectrum antibiotic that can inhibit most Gram-positive bacteria and is widely used in human medicine, breeding, animal husbandry and other fields.

[0003] Lincomycin is mainly produced by fermentation of Streptomyces lincolnensis. Currently, the strains used for industrial production of lincomycin are mainly mutants obtained by physical or chemical mutagenesis. However, traditional mutagenesis technology is time-consuming and labor-intensive, and has a high degree of randomness, and cannot ensure the efficient and stable production of lincomycin. Summary of the invention

[0004] In view of the above problems, the present invention provides a method for increasing the yield of lincomycin by knocking out the pA_gene0296 gene of Streptomyces lincomycin. The present invention can significantly increase the fermentation yield of lincomycin by knocking out the pA_gene0296 gene encoding the TetR / AcrR repressor protein in Streptomyces lincomycin.

[0005] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical scheme:

[0006] The first aspect of the present invention provides a method for improving lincomycin production by knocking out the pA_gene0296 gene of Streptomyces lincomycin: the pA_gene0296 gene of Streptomyces lincomycin is knocked out, and the resulting mutant is used to ferment and produce lincomycin, wherein the nucleotide sequence of the pA_gene0296 gene is shown in SEQ ID NO.1.

[0007] TetR / AcrR repressor protein encoding gene pA_gene0296 nucleotide sequence (SEQ ID NO.1):

[0008] .

[0009] TetR family transcriptional regulators (TetR family transcriptional regulators, TFRs) is a family of transcriptional regulators with the largest number of research findings. It can be used as a global regulatory factor, and can also be used to regulate the biosynthesis of antibiotics by pathway specificity. The AcrR of the TetR / AcrR family repressor protein is a multidrug binding protein of Escherichia coli. The protein is usually combined with an incomplete palindromic sequence in the upstream region of its own gene or the intergenic region between the repressor protein and the regulatory gene. This interaction inhibits RNA polymerase binding or blocks transcription initiation events to inhibit gene expression. The present invention has been found through research that the fermentation yield of lincomycin can be significantly increased by knocking out the TetR / AcrR repressor protein encoding gene pA_gene0296 gene in Linco Streptomyces. Prior art has not reported that knocking out the TetR / AcrR repressor protein encoding gene pA_gene0296 in Linco Streptomyces can significantly increase the yield of lincomycin.

[0010] The second aspect of the present invention provides a method for constructing a pA_gene0296 gene-deficient mutant of Streptomyces lincomyces: the pA_gene0296 gene in Streptomyces lincomyces is knocked out by genetic engineering; wherein the nucleotide sequence of the pA_gene0296 gene is shown in SEQ ID NO.1.

[0011] Preferably, the construction method uses Streptomyces lincomyces NBRC13054 as the starting strain.

[0012] Further preferably, the construction method specifically comprises the following steps:

[0013] S1. The nucleotide sequences shown in SEQ ID NO.2 and SEQ ID NO.3 are used as gRNA, and the PAM sequence is removed and then inserted into the promoter element skeleton, and a BbsⅠ restriction site is added, and the gRNA is connected to the pUC57 plasmid to obtain the pUC57-gRNA plasmid;

[0014] Using the genomic DNA of Streptomyces lincomyces NBRC13054 as a template, and using TetR-UF and TetR-UR as well as TetR-DF and TetR-DR as primers, respectively, the upstream and downstream homology arms of the pA_gene0296 gene were amplified by PCR reaction, wherein the nucleotide sequences of TetR-UF, TetR-UR, TetR-DF and TetR-DR are shown in SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7, respectively; the upstream and downstream homology arms amplified by PCR were connected by overlapping extension PCR method to obtain overlapping homology arms;

[0015] S2. Enzyme digestion of the pUC57-gRNA plasmid was performed, and the obtained enzyme digestion product was connected with the pCRISPomyces-2-RecA plasmid to obtain pCRISPomyces-2-RecA-gRNA; the pCRISPomyces-2-RecA plasmid was a plasmid obtained by adding a RecA overexpression gene cassette and an atpD overexpression gene cassette of the ATP synthase β subunit gene from Streptomyces coelicolor to the original pCRISPomyces-2 plasmid;

[0016] S3, the pCRISPomyces-2-RecA-gRNA is subjected to XbaⅠ single enzyme digestion, the obtained digestion product is dephosphorylated, and then connected with the homology arm of the overlap described in S1 to obtain the pC-TetR-H plasmid;

[0017] S4, transforming the pC-TetR-H plasmid into competent Escherichia coli cells, and culturing them as donor strains;

[0018] S5. The donor strain and mycelium of Streptomyces lincomyces are co-cultured in an antibiotic-free culture medium. After aerosol-like bacteria are formed, they are covered with antibiotics and cultured until conjugates grow. The pA_gene0296 gene-deficient Streptomyces lincomyces mutant strain is obtained through screening.

[0019] The above steps use genetic engineering technology to transform the coding gene of Streptomyces lincomycin, so that the pA_gene0296 gene is knocked out, thereby obtaining a high-yield engineered strain of lincomycin, that is, a pA_gene0296 gene-deficient mutant of Streptomyces lincomycin. The mutant can be used to efficiently produce lincomycin or a product in its synthesis process.

[0020] In this construction method, the primers used to amplify the upstream and downstream homologous arms of the pA_gene0296 gene are prone to non-specific bands during PCR amplification, and there are mixed bands around the target band. The above-mentioned TetR-UF, TetR-UR, TetR-DF and TetR-DR are obtained after multiple designs and have good specificity.

[0021] Preferably, the reaction system of PCR in S1 is:

[0022]

[0023] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 70°C for 30 s, extension at 72°C for 1 min, 35 cycles; and final extension at 72°C for 5 min.

[0024] Preferably, the small cycle reaction system of the overlap extension PCR method in S1 is:

[0025]

[0026] The reaction conditions for the minicycle were: 95°C for 3 min, 95°C for 30 s, 1 min according to the primer annealing temperature, 72°C for 1 min, 72°C for 5 min, and storage at 4°C for 5 cycles;

[0027] The macrocycle reaction system of the overlap extension PCR method described in S1 is:

[0028]

[0029] The macrocycle reaction conditions were: 95°C for 3 min, 95°C for 30 s, according to the primer annealing temperature for 1 min, 72°C for 1 min, 72°C for 5 min, and storage at 4°C for 35 cycles.

[0030] Preferably, the endonuclease used to digest the pUC57-gRNA plasmid in S2 is BbsⅠ.

[0031] Further preferably, the reaction system of the enzyme digestion is:

[0032]

[0033] The reaction conditions of the enzyme digestion are: 37° C. for 3 h and 65° C. for inactivation.

[0034] Preferably, the gRNA digested fragment is connected to the pCRISPomyces-2-RecA plasmid using the Golden Gate assembly method in S2.

[0035] Further preferably, the reaction system of the Golden Gate assembly is:

[0036]

[0037] The reaction program was: 37°C for 10 min, 16°C for 10 min, 9 cycles; 50°C for 5 min; 65°C for 20 min.

[0038] In the above enzyme ligation reaction system, the amount and ratio of each component are crucial to whether the ligation can be successful. Inappropriate addition ratio will cause ligation failure.

[0039] Preferably, the reaction system of the XbaⅠ single enzyme digestion reaction in S3 is:

[0040]

[0041] The reaction procedure was as follows: enzyme digestion at 37°C for 3 h and inactivation at 65°C.

[0042] Preferably, the dephosphorylation reaction system in S3 is:

[0043]

[0044]

[0045] The reaction procedure was: 37°C for 10 min, 80°C for 2 min.

[0046] Preferably, Clon is used in S3 IIOne Step Cloning Kit C112 enzyme ligates the dephosphorylated product to the homology arm of the overlap.

[0047] Further preferably, the reaction system of the connection is:

[0048]

[0049] The reaction conditions were: 30 min at 37°C

[0050] Preferably, the Escherichia coli competent cells in S4 are ET12567 Escherichia coli competent cells; and the Streptomyces lincomyces in S5 is Streptomyces lincomyces NBRC13054.

[0051] Preferably, the specific operation method of S5 is: mixing the bacterial suspension of the donor strain with the mycelial suspension of Streptomyces lincomyces, centrifuging, discarding the supernatant, culturing the bacterial liquid on an antibiotic-free WL-50 plate until a mist of bacteria is formed on the surface, covering it with antibiotics, and culturing it inverted until conjugates grow, and obtaining a pA_gene0296 gene-deficient Streptomyces lincomyces mutant strain after screening.

[0052] Further preferably, the antibiotics are apramycin and sodium nalidixate, which are covered in the form of a solution, dried after covering, and then inverted for culture. Further preferably, the volume of the solution of apramycin and sodium nalidixate is 10 times the volume of the bacterial solution, the concentration of the apramycin solution is 1 mg / mL, and the concentration of the sodium nalidixate solution is 0.5 mg / mL. The dosage and concentration of the two can ensure that they have resistance and can be used for covering.

[0053] The third aspect of the present invention provides a pA_gene0296 gene-deficient mutant strain of Streptomyces lincomyces constructed by the above construction method.

[0054] The fourth aspect of the present invention provides the use of the pA_gene0296 gene-deficient mutant of Streptomyces lincomycin in the fermentation production of lincomycin.

[0055] Preferably, the pA_gene0296 gene-deficient mutant of Streptomyces lincomyces is first cultured in a primary fermentation medium for 2 to 4 days at a fermentation temperature of 28°C, and then inoculated into a secondary fermentation medium for fermentation and culture for 6 to 8 days at a fermentation temperature of 28°C; the components of the primary fermentation medium are: 10 g / L glucose, 20 g / L soluble starch, 10 g / L soybean cake powder, 3 g corn steep liquor, 4 g / L CaCO3, 1.5 g / L (NH4)2SO4, and pH = 7.0-7.2; the components of the secondary fermentation medium are: 100 g / L glucose, 20 g / L soybean cake powder, 1.5 g / L (NH4)2SO4, 1.5 g / L corn steep liquor, 8 g / L NaNO3, 0.3 g / L K2HPO3, 8 g / L CaCO3, and pH = 7.0-7.2.

[0056] The beneficial effect of the present invention is that the present invention significantly improves the lincomycin yield by knocking out the TetR / AcrR regulatory factor pA_gene0296 gene near the lincomycin biosynthesis gene cluster. The results show that after the TetR / AcrR repressor protein is missing, the average lincomycin yield in the lincomycin yield fermentation broth of the obtained mutant is significantly higher than the yield of the wild-type lincomycin Streptomyces. The construction method of the pA_gene0296 gene-deficient lincomycin Streptomyces provided by the present invention is to transform the coding gene of lincomycin Streptomyces by genetic engineering technology, thereby obtaining a lincomycin high-yield engineered strain, and the obtained engineered strain can be used for the efficient production of lincomycin or the product in its synthesis process. The experiment verified that the average lincomycin yield in the fermentation broth of pA_gene0296 gene-deficient Streptomyces lincomycin obtained by the construction method can reach 145.42 mg / L, which is 115.50 mg / L of lincomycin in the fermentation broth of the wild-type NBRC13054 strain. The yield of lincomycin produced by fermentation of pA_gene0296 gene-knockout Streptomyces lincomycin is increased by 25.91% compared with the wild-type strain. The present invention provides a new method for increasing the yield of lincomycin biosynthesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 This is a schematic diagram of the structure of pCRISPomyces-2-RecA in Example 1;

[0058] Figure 2 This is a schematic diagram of the target gRNA inserted into the promoter element skeleton in Example 1;

[0059] Figure 3 The electrophoresis diagram of the upstream and downstream homology arms of the pA_gene0296 gene after PCR amplification in Example 1; M: DL15000+2000Marker; 1: upstream homology arm; 2: downstream homology arm;

[0060] Figure 4 This is the electrophoresis diagram after overlap PCR of the upstream and downstream homology arms of the pA_gene0296 gene in Example 1;

[0061] Figure 5 The electrophoresis diagram of the pUC57-TetR-sg plasmid after BbsⅠ digestion in Example 1; M: DL15000+2000Marker;

[0062] Figure 6 The positive clone verification result of pC-TetR-H plasmid in Example 1;

[0063] A: pC-TetR-H plasmid PCR verification results; M: Marker 1Kb Ladder; 1-2: positive clones;

[0064] B: pC-TetR-H plasmid restriction enzyme digestion verification results; M: DL150000+2000Marker; 1-2: positive clones;

[0065] Figure 7 This is a schematic diagram of the construction of the editing plasmid in Example 1;

[0066] Figure 8 The pA_gene0296 gene deletion strain screening results in Example 2;

[0067] A: initial screening results; M: Marker 1Kb; Ladder 1-9: screening strains

[0068] B: Secondary verification result; M: Marker 1Kb; Ladder 1-6: Corresponding to A Figure 3 , 4 , 6, 7, 8, 9 pipes;

[0069] Fig. 9 The PCR verification results of the ΔTetR mutant in Example 2; M: Marker 1Kb; Ladder: 1: wild-type strain; 2: ΔTetR strain;

[0070] Fig.10 The results of pC-TetR-H plasmid elimination in Example 2; the left picture is an Apr-resistant WL-50 plate; the right picture is a non-resistant WL-50 plate;

[0071] Fig.11 The growth curves of the ΔTetR mutant and the wild-type strain NBRC13054 in Example 2;

[0072] Fig.12 It is the lincomycin standard curve in Example 3;

[0073] Fig.13 The HPLC chromatogram of lincomycin standard in Example 3;

[0074] Fig.14 HPLC graphs of the ΔTetR mutant and NBRC13054 strain in Example 3;

[0075] Fig.15 This is a bar graph of lincomycin production by the ΔTetR mutant and the NBRC13054 strain in Example 3. DETAILED DESCRIPTION

[0076] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work fall within the scope of protection of the present invention.

[0077] Lincomycin is produced by Streptomyces lincomycin and is a broad-spectrum antibiotic widely used to treat infectious diseases caused by Gram-positive bacteria. The market demand is growing, so it is particularly important to increase its production. However, the strains that currently biosynthesize lincomycin are mainly obtained through physical or chemical mutagenesis methods. Mutagenesis technology is time-consuming, labor-intensive and has a high degree of randomness. It cannot ensure the efficient and stable production of lincomycin, nor can it provide theoretical guidance for the precise and efficient breeding of high-yield lincomycin.

[0078] To address this problem, an embodiment of the present invention provides a method for increasing the yield of lincomycin by knocking out the pA_gene0296 gene of Streptomyces lincomycin: knocking out the pA_gene0296 gene of Streptomyces lincomycin, and using the resulting strain to ferment and produce lincomycin. TetR / AcrR repressor protein can inhibit lincomycin synthesis, and the pA_gene0296 gene is a gene encoding the TetR / AcrR repressor protein. This method deletes the gene to cause the TetR / AcrR repressor protein to be missing, thereby significantly increasing the fermentation yield of lincomycin.

[0079] The embodiment of the present invention also provides a method for constructing a pA_gene0296 gene-deficient mutant strain of Streptomyces lincomyces.

[0080] The embodiment of the present invention also provides a pA_gene0296 gene-deficient mutant strain of Streptomyces lincomyces constructed by the above construction method.

[0081] The present invention also provides the use of the pA_gene0296 gene-deficient lincomycin mutant strain in the fermentation production of lincomycin.

[0082] The scheme of the present invention is described below through specific embodiments.

[0083] The experimental strains and plasmids used in the following examples are shown in Table 1.

[0084] Table 1 Experimental strains and plasmids

[0085]

[0086] The culture medium used in the following examples is as follows:

[0087] LB liquid medium: yeast extract 5g / L, tryptone 10g / L, sodium chloride 10g / L;

[0088] WL-50 medium: yeast extract 1g / L, soluble starch 5g / L, sodium chloride 2.5g / L, magnesium sulfate 3.6g / L, L-alanine 0.2g / L, L-arginine 0.2g / L, L-asparagine 0.5g / L, magnesium chloride 4.8g / L;

[0089] ISP-2 medium: malt extract 10 g / L, yeast extract 4 g / L, glucose 4 g / L, peptone 4 g / L, pH = 7.3;

[0090] 2×YT medium was purchased from Qingdao Hi-Tech Park Haibo Biotechnology Co., Ltd., product number HBDC003;

[0091] Primary fermentation medium: glucose 10g / L, soluble starch 20g / L, soybean cake powder 10g / L, corn steep liquor 3g, CaCO3 4g / L, (NH4)2SO4 1.5g / L, pH=7.0-7.2;

[0092] Secondary fermentation medium: glucose 100 g / L, soybean cake powder 20 g / L, (NH4)2SO4 1.5 g / L, corn steep liquor 1.5 g / L, NaNO3 8 g / L, K2HPO3 0.3 g / L, CaCO3 8 g / L, pH = 7.0-7.2.

[0093] The endonucleases used in the following examples were purchased from NEB.

[0094] Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art; the materials, reagents, etc. used in the following examples can be obtained from commercial channels.

[0095] Example 1

[0096] This example provides a process for constructing a TetR / AcrR repressor protein knockout vector.

[0097] 1. Design of guideRNA (gRNA)

[0098] The RecA overexpression gene cassette and the atpD overexpression gene cassette from the ATP synthase β subunit gene from Streptomyces coelicolor were added to the original pCRISPomyces-2 plasmid to obtain the pCRISPomyces-2-RecA plasmid, the structure of which is shown in the figure below: Figure 1 shown.

[0099] Based on the successful transformation of pCRISPomyces-2-RecA plasmid, a knockout vector of the TetR / AcrR negative regulatory factor encoding gene pA_gene0296 was constructed.

[0100] The PAM sequence corresponding to the Cas9 protein of the pCRISPomyces-2 plasmid is NGG. The CHOPCHOP website (http: / / chopchop.cbu.uib.no / ) was used to design the gRNA for the target gene TetR / AcrR sequence of Streptomyces lincomycetes NBRC13054. Two gRNAs with high scores and no mismatches were selected, 5'-CCTGCACATCGACCGCCCGA-3' (gRNA1) and 5'-CGTCGAGACCACGATCACGC-3' (gRNA2), as the target gRNAs. After obtaining the target gRNA, the PAM sequence was removed to prevent the plasmid from self-cleavage and inserted into the constructed promoter element skeleton (such as Figure 2 As shown, the green part is the site for inserting the target gRNA), a BbsⅠ restriction site was added, and it was synthesized by Qingke Biotechnology Company and connected to the pUC57 plasmid, named pUC57-gRNA.

[0101] 2. Obtaining homology arms

[0102] Using the genomic DNA of Streptomyces lincomyces NBRC13054 as a template, the 1000bp sequences of the TetR / AcrR negative regulatory factor encoding gene pA_gene0296 were selected as the upstream and downstream homology arms, and the primers TetR-UF / TetR-UR and TetR-DF / TetR-DR were designed, where the 5' ends of the upstream homology arm primer TetR-UF and the downstream homology arm primer TetR-DR were added with XbaⅠ restriction sites, and the upstream and downstream homology arm fragments of the target gene were amplified by PCR reaction. The primer sequences are shown in Table 2, the PCR reaction system is shown in Table 3, and the PCR reaction conditions are as follows: 95℃ pre-denaturation for 3min; 95℃ denaturation for 30s, 70℃ annealing for 30s, 72℃ extension for 1min, 35 cycles; 72℃ final extension for 5min.

[0103] Table 2 Primers for gene sequence amplification

[0104]

[0105] Table 3 Homologous arm PCR reaction system

[0106]

[0107] The upstream and downstream homology arm PCR amplification products were subjected to agarose gel electrophoresis. Figure 3As shown. Then, after gel recovery of the upstream and downstream homology arms, the upstream and downstream homology arms were connected using the overlap extension PCR method (Overlap PCR). Mini-cycle reaction program: 95℃3min, 95℃30s, according to the primer annealing temperature for 1min, 72℃1min, 72℃5min, 4℃ storage, 5 cycles. The mini-cycle reaction system is shown in Table 4.

[0108] The macrocycle used the microcycle reaction solution as a template, and the program was: 95°C for 3 min, 95°C for 30 s, 1 min according to the primer annealing temperature, 72°C for 1 min, 72°C for 5 min, and 4°C storage, for 35 cycles. The macrocycle reaction system is shown in Table 5.

[0109] Table 4 Overlap extension PCR mini-cycle reaction system

[0110]

[0111] Table 5 Overlap extension PCR macrocycle reaction system

[0112]

[0113] The agarose gel electrophoresis results of the upstream and downstream homology arms after ligation are as follows Figure 4 Gel recovery and purification were performed for plasmid ligation.

[0114] 3. Guide RNA ligation

[0115] Since there are too many BbsⅠ sites on the genomic DNA of Streptomyces lincomyces, in order to prevent plasmid fragmentation during BbsⅠ digestion after connecting the homology arms, this experiment first performed BbsⅠ digestion reaction on the pUC57-gRNA plasmid. The reaction procedure was: 37℃ digestion for 3h, 65℃ enzyme inactivation for 10min, and 4℃ storage. The digestion system is shown in Table 6:

[0116] Table 6 Single enzyme digestion reaction system

[0117]

[0118]

[0119] The digested product and the original plasmid were subjected to agarose gel electrophoresis at the same time. The electrophoresis diagram is shown in Figure 5 As shown. The 499 bp band of the gRNA fragment cleaved by enzyme was recovered and purified by gel purification.

[0120] The pCRISPomyces-2-RecA plasmid was extracted, and the gRNA enzyme fragment was connected to the pCRISPomyces-2-RecA plasmid using the Golden Gate assembly method. After successful connection, it was named pCRISPomyces-2-RecA-gRNA and transformed into the Top10 competent medium. The Golden Gate assembly reaction system is shown in Table 7.

[0121] Table 7 Golden Gate assembly reaction system

[0122]

[0123] The reaction procedure was: 37°C for 10 min, 16°C for 10 min, 9 cycles; 50°C for 5 min; 65°C for 20 min; and storage at 4°C.

[0124] 4. Plasmid digestion

[0125] The pCRISPomyces-2-RecA-gRNA plasmid was extracted and subjected to XbaⅠ single restriction enzyme digestion reaction. The reaction procedure was 37℃ restriction enzyme digestion for 3h, 65℃ inactivation, and 4℃ storage. The restriction enzyme digestion product was subjected to agarose gel electrophoresis, and then the target band was recovered using the OMEGA gel recovery kit. The restriction enzyme digestion system is shown in Table 8:

[0126] Table 8 Single enzyme digestion reaction system

[0127]

[0128] After single restriction digestion, the pCRISPomyces-2-RecA-gRNA plasmid is prone to self-ligation. Before ligating with the homology arm of overlap, it needs to be dephosphorylated with CIP. The reaction procedure is 37℃ for 10min and 80℃ for 2min. The reaction system is shown in Table 9:

[0129] Table 9 CIP dephosphorylation system

[0130]

[0131] 5. Homology arm connection

[0132] Using Clon IIOne Step Cloning Kit C112 enzyme, the dephosphorylated pCRISPomyces-2-RecA-gRNA was ligated to the homology arms of overlap at 37°C for 30 min. The reaction system is shown in Table 10.

[0133] Table 10C112 Ligase Enzyme Reaction System

[0134]

[0135] The ligated product was directly transformed into E. coli Top10, spread on LB (Apr resistance) plates, and cultured overnight in a 37°C incubator until a single colony grew.

[0136] A single colony was picked and expanded in 40 mL LB liquid medium and plasmid DNA was extracted. The positive colony was about 3300 bp, as shown in Table 11. Figure 6 As shown in Figure A; XbaⅠ enzyme was used for restriction digestion verification, and approximately 2000bp upstream and downstream homology arm bands were obtained, as shown in Figure 6 As shown in Figure B; and sent to Sangon Biotechnology (Shanghai) Co., Ltd. for sequencing, the constructed pC-TetR-H plasmid was stored in a -80°C refrigerator with glycerol.

[0137] Table 11 JC-F / JC-R primers

[0138]

[0139]

[0140] The schematic diagram of the construction of the pC-TetR-H plasmid is as follows Figure 7 shown.

[0141] Example 2

[0142] This example provides a Streptomyces lincomyces ΔTetR in which the gene pA_gene0296 encoding the TetR / AcrR repressor protein is knocked out.

[0143] 1. Conjugation transfer between Streptomyces lincomyces and Escherichia coli

[0144] The pC-TetR-H plasmid was transformed into ET12567 (pUZ8002) Escherichia coli competent cells, and a single colony was picked for PCR verification using JC-F / JC-R primers. After successful verification, conjugation transfer was performed with Streptomyces lincomyces NBRC13054:

[0145] Donor strain: Inoculate ET12567 E. coli competent cells containing pC-TetR-H plasmid into 50mL LB medium, shake at 37℃ for 3-4h. Take 5mL of bacterial solution and centrifuge to remove the supernatant. Add 5mL LB, vortex, centrifuge to remove the supernatant, repeat twice. Finally, add 500μL LB and mix well by pipetting.

[0146] Recipient strain: Spread Streptomyces lincomyces NBRC13054 on a WL-50 solid plate. Scrape an appropriate amount of spores and inoculate them into ISP-2 medium, culture at 28°C for 2-3 days. Observe whether the mycelium of Streptomyces lincomyces is finely and evenly dispersed in the ISP-2 medium. Take 5mL of bacterial solution and centrifuge to remove the supernatant; then add an equal volume of 10% glycerol, centrifuge to remove the supernatant, and wash the mycelium; then add an equal volume of 2×YT medium, centrifuge to remove the supernatant, and repeat the washing twice; finally, add 500μL of 2×YT to resuspend and mix thoroughly.

[0147] Vortex 500 μL of mycelium and 500 μL of ET12567 competent E. coli cells containing pC-TetR-H plasmid, and centrifuge to remove part (200 μL) of supernatant. Pipette 100 μL of the mixed bacterial solution and spread it on a non-antibiotic WL-50 plate, and culture it in a 28°C incubator until a layer of mist-like bacteria grows on the WL-50 plate. Pipette 1 mL of a mixed antibiotic aqueous solution containing 0.5 mg / mL sodium nalidixic acid and 1 mg / mL apramycin to cover it, dry it completely in the clean bench, and then invert it and place it in a 28°C incubator to culture until conjugates grow.

[0148] 2. Screening of strains lacking TetR / AcrR repressor protein

[0149] The pC-TetR-H plasmid was successfully transferred by conjugation. After the conjugates were subcultured, several single colonies that could produce spores were picked, and genomic DNA was extracted from 9 strains in each group. PCR verification was performed using the detection primers TetR-F / TetR-R (as shown in Table 12) and compared with the wild-type genome. The detection primers TetR-F / TetR-R need to be designed at 500 bp outside the upstream and downstream homology arms used to construct the pC-TetR-H plasmid.

[0150] Table 12 ΔTetR strain screening and detection primers

[0151]

[0152] PCR verification was performed on the 9 strains of the first group. The engineered strain that successfully knocked out the TetR / AcrR repressor protein encoding gene pA_gene0296 should have a CDS region 594 bp shorter than the wild type, and the PCR product was 3000 bp. The wild type PCR product was 3500 bp. The verification found that each pipeline had a 3000 bp band, but only the bands of pipelines 3, 4, 6, 7, 8, and 9 were single, such as Figure 8 As shown in Figure A. The pipeline PCR was verified again and it was found that the bands were single and all were 3000 bp. Figure 8 As shown in Figure B. Compared with the wild type, it is proved that the TetR / AcrR repressor protein gene fragment has been successfully knocked out, as shown in Figure Fig. 9Finally, the fragments were recovered and the strains were sent to Bioengineering for sequencing. The sequencing results showed that the TetR / AcrR repressor protein encoding gene pA_gene0296 in Streptomyces lincosus was successfully knocked out, and the successfully transformed strain was named ΔTetR strain.

[0153] 3. Plasmid elimination

[0154] The pCRISPomyces-2-RecA plasmid is a thermosensitive plasmid that contains a high temperature condition-deficient replication initiation site pSG5 and does not replicate at 37°C. The successfully transferred conjugates were cultured on a non-resistance plate at 37°C for three consecutive generations to cause the loss of the pC-TetR-N and pC-TetR-H plasmids, thereby achieving a seamless knockout of the TetR / AcrR repressor protein encoding gene pA_gene0296. The pA_gene0296 gene knockout strain that had been subcultured multiple times at 37°C was cultured on a conidial plate without resistance at 28°C. After spores grew, spores from the same single colony were picked and cultured on a plate containing 25 μg / mL Amp and a conidial plate without resistance as controls. The results are shown in the figure. Fig.10 As shown, the conjugates can grow normally and produce spores on a plate without apramycin, but cannot grow normally on a resistant plate, indicating that the pC-TetR-H plasmid was successfully eliminated.

[0155] 4. Growth curve determination of ΔTetR mutant strain

[0156] After the pC-TetR-H plasmid was eliminated from the ΔTetR mutant, it was inoculated with the lincomycin NBRC13054 strain in ISP-2 liquid medium for activation, and then spread on WL-50 solid medium. After spore production, 1 cm 2 The spore culture medium was inoculated with 40 mL of ISP-2 liquid medium and cultured in a shake flask for 36 h, and then cultured in a shake flask at 28 °C and 280 r / min for 7 days. From day 0, samples were taken every 12 h, with 3 biological replicates each time. After all the cells in the culture medium were filtered out, they were dried in a 50 °C oven, the dry weight of the cells was weighed, and a growth curve was drawn.

[0157] like Fig.11As shown, the ΔTetR mutant and the wild-type NBRC13054 strain were both in the logarithmic phase of cell growth from 0 to 48 h, and the difference in the increase in dry weight of mycelium was small; the number of viable cells in the stable period of microbial cells remained relatively stable, and the bacterial mass was able to reach the highest level, which was a period of nutrient consumption and accumulation of fermentation products. By analyzing the growth curves of the two strains, it can be seen that the mycelium dry weight of the wild-type NBRC13054 strain reached the maximum value at 48h, and the mycelium dry weight at 60h dropped sharply, while the mycelium dry weight of the ΔTetR mutant remained relatively stable during 48-84h, indicating that the ΔTetR mutant was in a stable period at 48-84h, and the duration of the stable period was longer than that of the wild-type NBRC13054 strain. The longer duration of the stable period of the ΔTetR mutant may be more conducive to the production and accumulation of metabolites such as antibiotics, endotoxins, and exotoxins; at the same time, it was found that the mycelium dry weight of the ΔTetR mutant was higher than that of the wild strain at 3.04g / L, reaching 3.913g / L. The above results show that the TetR / AcrR regulatory factor has an effect on the bacterial growth of Streptomyces lincosus NBRC13054, and to a certain extent inhibits the transcription and expression of related genes during the growth of the bacteria.

[0158] Example 3

[0159] This embodiment provides a method for increasing the yield of lincomycin by using a ΔTetR mutant strain of Streptomyces lincomycin in which the pA_gene0296 gene is knocked out.

[0160] The wild strain of Streptomyces lincomyces and the ΔTetR mutant strain with the pC-TetR-H plasmid eliminated were cultured on WL-50 solid plates at 30°C for 7 days. 2 The bacterial block was cultured in the seed culture medium for 48 hours, and then inoculated into the primary fermentation medium at a 2% inoculation rate, and fermented in a shake flask at 28°C and 280 r / min for 3 days. Then, the inoculation rate was inoculated into the secondary fermentation medium at a 2% inoculation rate, and fermented in a shake flask at 28°C and 280 r / min for 7 days.

[0161] Lincomycin detection: After the fermentation is completed, 2 mL of fermentation liquid is taken, and the supernatant is taken after centrifugation. Then 1.5 times the volume of anhydrous ethanol is added, and the supernatant after centrifugation is filtered with a 0.22 μm organic phase filter membrane. The treated fermentation liquid is detected by HPLC, and the detection conditions are: the chromatographic column uses Agilent ZORBAX SB-C18 (4.6×250mm 5μm), the detection wavelength is 210nm, the mobile phase is 50mmol / L ammonium acetate (pH=9) and methanol (volume ratio is 20:80), the flow rate is 0.4mL / min, the injection volume is 10μL, and the liquid chromatography instrument is Agilent 1260 high performance liquid chromatograph.

[0162] Standard curve drawing: Weigh a certain amount of lincomycin standard, prepare 10 mg / mL lincomycin stock solution, and then dilute it to 25 mg / L, 50 mg / L, 100 mg / L, 300 mg / L, 500 mg / L in sequence. According to the lincomycin liquid phase detection conditions, measure the peak area of ​​each concentration standard, and fit the standard curve with lincomycin concentration as the horizontal axis and peak area as the vertical axis, as shown in the figure. Fig.12 The obtained linear regression equation is: y = 5.6688x + 89.27, R 2 =0.9961.

[0163] The peak time of lincomycin standard is 8min 50s. Fig.13 The chromatogram of lincomycin after the fermentation liquid of ΔTetR mutant and wild strain NBRC13054 was diluted 5 times with ethanol is shown in Fig.14 As shown, the results were put into the regression equation to calculate the lincomycin production of the mutant strain compared with the wild strain. Fig.15 shown.

[0164] The results showed that the average lincomycin production in the fermentation broth of the ΔTetR mutant strain could reach 145.42 mg / L, while the average lincomycin production in the fermentation broth of the wild-type strain was 115.50 mg / L. The lincomycin production after the TetR / AcrR repressor protein was knocked out increased by 25.91% compared with the wild-type, and the loss of the TetR / AcrR repressor protein significantly increased the lincomycin production, indicating that the TetR / AcrR regulatory factor pA_gene0296 near the lincomycin biosynthetic gene cluster of Streptomyces lincomycin can inhibit the production of lincomycin.

[0165] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for increasing lincomycin production by knocking out the pA_gene0296 gene of Streptomyces lincomycin, characterized in that: The pA_gene0296 gene of Streptomyces lincomycin was knocked out, and the obtained mutant was used to ferment and produce lincomycin. The nucleotide sequence of the pA_gene0296 gene is shown in SEQ ID NO.

1.

2. A method for constructing a pA_gene0296 gene-deficient mutant of Streptomyces lincomyces, characterized in that: The pA_gene0296 gene in Streptomyces lincomyces was knocked out by genetic engineering; wherein the nucleotide sequence of the pA_gene0296 gene is shown in SEQ ID NO.

1.

3. The construction method according to claim 2, characterized in that: The construction method uses Streptomyces lincomyces NBRC13054 as the starting strain.

4. The construction method according to claim 2 or 3, characterized in that: The specific steps include: S1. The nucleotide sequences shown in SEQ ID NO.2 and SEQ ID NO.3 are used as gRNA, and the PAM sequence is removed and then inserted into the promoter element skeleton, and a BbsⅠ restriction site is added, and the gRNA is connected to the pUC57 plasmid to obtain the pUC57-gRNA plasmid; Using the genomic DNA of Streptomyces lincomyces NBRC13054 as a template, TetR-UF and TetR-UR as well as TetR-DF and TetR-DR as primers, the upstream and downstream homology arms of the pA_gene0296 gene were amplified by PCR reaction, respectively. The nucleotide sequences of TetR-UF, TetR-UR, TetR-DF and TetR-DR are shown in SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7, respectively. The upstream and downstream homology arms obtained by PCR amplification are connected by overlapping extension PCR method to obtain overlapping homology arms; S2. Enzyme digestion of the pUC57-gRNA plasmid was performed, and the obtained enzyme digestion product was connected with the pCRISPomyces-2-RecA plasmid to obtain pCRISPomyces-2-RecA-gRNA; the pCRISPomyces-2-RecA plasmid was a plasmid obtained by adding a RecA overexpression gene cassette and an atpD overexpression gene cassette of the ATP synthase β subunit gene from Streptomyces coelicolor to the original pCRISPomyces-2 plasmid; S3, the pCRISPomyces-2-RecA-gRNA is subjected to XbaⅠ single enzyme digestion, the obtained digestion product is dephosphorylated, and then connected with the homology arm of the overlap described in S1 to obtain the pC-TetR-H plasmid; S4, transforming the pC-TetR-H plasmid into competent Escherichia coli cells, and culturing them as donor strains; S5. The donor strain and mycelium of Streptomyces lincomyces are co-cultured in an antibiotic-free culture medium. After aerosol-like bacteria are formed, they are covered with antibiotics and cultured until conjugates grow. The pA_gene0296 gene-deficient Streptomyces lincomyces mutant strain is obtained through screening.

5. The construction method according to claim 4, characterized in that: The endonuclease used to digest the pUC57-gRNA plasmid in S2 is BbsⅠ.

6. The construction method according to claim 4, characterized in that: In S2, the gRNA digestion fragment was connected to the pCRISPomyces-2-RecA plasmid using the Golden Gate assembly method.

7. The construction method according to claim 4, characterized in that: Using Clon in S3 II One Step Cloning Kit C112 enzyme ligates the dephosphorylated product to the homology arms of the overlap.

8. The construction method according to claim 4, characterized in that: The E. coli competent cells described in S4 are ET12567 E. coli competent cells; the Streptomyces lincomyces described in S5 are Streptomyces lincomyces NBRC13054.

9. A pA_gene0296 gene-deficient mutant of Streptomyces lincomyces constructed by the construction method according to any one of claims 2 to 8.

10. Use of the pA_gene0296 gene-deficient mutant of Streptomyces lincomycin according to claim 9 in the fermentation production of lincomycin.

Images