Genetic engineering strain for producing mupirocin as well as preparation method and application thereof

Abstract

The invention provides a gene engineering strain for producing mupirocin and a preparation method thereof. The rsmA is found to be a negative regulation gene for the first time, that is to say, the yield of mupirocin of pseudomonas fluorescens can be increased by knocking out the rsmA gene and reducing the expression of the rsmA gene or reducing the activity of rsmA protein. Furthermore, pseudomonas fluorescens is taken as a chassis strain, regulatory genes rsmA, rsmN and xylB genes are knocked out, and mupR genes are respectively integrated at corresponding positions, so that the strain for producing mupirocin is obtained; wherein rsmA is a negative regulation site, and rsmN and xylB are neutral sites, which belong to genome natural sites, are relatively stable and are not easy to lose in a passage process; meanwhile, the rsmA is knocked out and replaced with the mupR gene, the mupirocin yield can be increased through mupR gene expression, the negative regulation effect of the rsmA can be eliminated, and the mupirocin yield can be increased in a synergistic mode.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering and relates to a genetically engineered strain for producing mupirocin and a preparation method and application thereof. Background Art

[0002] The polyketide antibiotic mupirocin is composed of pseudomononic acids (PA) A, B, C, and D, with pseudomononic acid A (PA-A) being its primary active ingredient. Mupirocin competitively inhibits the activity of the bacterial isoleucyl-tRNA synthase IleRS, thereby hindering isoleucine incorporation and inhibiting protein synthesis in sensitive bacteria, ultimately leading to bacterial death. As a result, it is highly effective and broadly effective against Gram-positive bacteria, such as Staphylococci and Streptococci. It exhibits excellent antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), making it the preferred topical antibiotic for the treatment of MRSA worldwide. Mupirocin also antagonizes some Gram-negative bacteria, such as Haemophilus influenzae, Neisseria meningitidis, and Neisseria gonorrhoeae; it also has a limited antibacterial effect against some fungal pathogens, such as Malassezia furfur, Candida albicans, and Trichophyton mentagrophytes. Because mupirocin is rapidly metabolized in the human body and has minimal toxic side effects, it has been widely used as a topical antibiotic in the treatment of skin diseases and in combination with other drugs to prevent and treat infections.

[0003] Mupirocin can be synthesized by microorganisms or chemically. The chemical synthesis method has many steps, a long synthesis route, and a low yield; the intermediates are unstable, requiring expensive catalysts, and the process is energy-intensive, resulting in high costs and environmental unfriendliness. The current mainstream method for synthesizing mupirocin is through microbial fermentation, whose enzymatic reactions efficiently assemble molecular skeletons, the fermentation process consumes less energy, and the product purity is high. The main problem in current fermentation production applications is the low fermentation production level of natural mupirocin production strains. Modifying production strains through molecular biology and other technical means to obtain high-yield fermentation strains is an effective means to increase industrial fermentation capacity.

[0004] Mupirocin was originally isolated and extracted from Pseudomonas fluorescens NCIMB 10586. Its biosynthetic proteins were found to be encoded by a 74 kb mup gene cluster. Its biosynthesis pathway is complex and strictly regulated by the quorum sensing (QS) system MupI / MupR within the cluster. MupR, a member of the LuxR family of quorum sensing regulatory genes, generally plays a positive role in regulating antibiotic biosynthesis. Studies have shown that deletion of the transcriptional regulator MupR and the signaling molecule synthase MupI genes in NCIMB 10586 significantly reduces mupirocin production, while overexpression of the mupR gene increases mupirocin production. However, when inserting the mupR gene into the Pseudomonas fluorescens genome, it is important to consider whether the inserted gene will affect the expression of adjacent genes to avoid polarizing effects. Therefore, the construction of stable genetically engineered strains requires careful selection of the insertion site. However, there are currently few reports on the insertion sites of the mupR gene in the genome of Pseudomonas fluorescens, resulting in problems such as low production capacity of natural mupirocin production bacteria. Summary of the Invention

[0005] To solve the above technical problems, the present invention aims to provide a genetically engineered strain for producing mupirocin and a preparation method thereof. The present invention studies the application of the rsmA gene in improving the mupirocin yield of Pseudomonas fluorescens, and for the first time finds that rsmA is a negative regulatory gene, that is, by knocking out the rsmA gene, reducing the expression of the rsmA gene, or reducing the activity of the rsmA protein to improve the mupirocin yield of Pseudomonas fluorescens. Further, the present invention uses Pseudomonas fluorescens as a chassis strain, by knocking out the regulatory genes rsmA, rsmN, and xylB genes, and integrating the mupR gene in the corresponding positions, to obtain the strain producing mupirocin; wherein rsmA is a negative regulatory site, rsmN and xylB are neutral sites, all of which belong to natural genomic sites, are relatively stable, and are not easily lost during passage; while knocking out rsmA and replacing it with the mupR gene can not only utilize the mupR gene expression to improve mupirocin yield, but also eliminate the negative regulatory effect of rsmA, and synergistically improve mupirocin yield. The fermentation yield of mupirocin by this genetically engineered bacterium reached 7,561 mg / L, of which the yield of pseudomonadic acid A was 7,060 mg / L, providing a solid foundation for its industrial fermentation production.

[0006] The purpose of the present invention can be achieved by the following solutions:

[0007] In a first aspect, the present invention provides an application of the rsmA gene in improving the mupirocin production of Pseudomonas fluorescens, wherein the nucleotide sequence of the rsmA gene is shown in SEQ ID NO.1;

[0008] The method for improving the mupirocin production of P. fluorescens comprises one or more of the following: knocking out the rsmA gene, reducing the expression of the rsmA gene, and reducing the activity of the rsmA protein.

[0009] As an embodiment of the present application, the P. fluorescens comprises the P. fluorescens NCIMB10586 strain. The strain preservation center name: National Collection of Industrial Food and Marine Bacteria (NCIMB), preservation address: NCIMB Ltd, Wellheads Place, Dyce, Aberdeen, AB217GB, UK. Preservation number: NCIMB 10586, preservation time: January 3, 1972.

[0010] In a second aspect, the present application provides the use of an inhibitor of the rsmA gene in improving the mupirocin production of P. fluorescens, wherein the inhibitor comprises one or more of the following: a reagent for knocking out the rsmA gene, a reagent for reducing the expression of the rsmA gene, and a reagent for reducing the activity of the rsmA protein.

[0011] In a third aspect, the present application provides a method for constructing a mupirocin-producing strain, wherein P. fluorescens is used as a chassis strain, the rsmA gene is knocked out and replaced with the mupR gene to obtain the mupirocin-producing strain.

[0012] In a fourth aspect, the present application provides a method for constructing a mupirocin-producing strain, wherein P. fluorescens is used as a chassis strain, the rsmA, rsmN and xylB genes are knocked out, and the mupR gene is integrated at the corresponding positions to obtain the mupirocin-producing strain. That is, P. fluorescens is used as a chassis strain, the rsmA, rsmN and xylB genes are knocked out, and the mupR gene is replaced to obtain the mupirocin-producing strain.

[0013] As an embodiment of the present application, the nucleotide sequence of the rsmN gene is shown in SEQ ID NO. 2; the nucleotide sequence of the xylB gene is shown in SEQ ID NO. 3; and the nucleotide sequence of the mupR gene is shown in SEQ ID NO. 4.

[0014] As an embodiment of the present application, the method for constructing specifically comprises the following steps:

[0015] S1, the method for knocking out the xylB gene and replacing the mupR is as follows:

[0016] S1.1. Amplify the upstream and downstream homology arms of the xylB gene (upstream and downstream primers are SEQ ID NO. 5 / SEQ ID NO. 6 and SEQ ID NO. 9 / SEQ ID NO. 10, respectively) and the tac promoter-mupR gene-terminator sequence (using primers SEQ ID NO. 7 / SEQ ID NO. 8), connect them using fusion PCR, and clone them into the pK18mobs acB suicide plasmid to obtain the recombinant plasmid pK18-BR;

[0017] S1.2. Transform the recombinant plasmid pK18-BR into E. coli S17 and co-culture it with the NCIMB 10586 strain. Use LB double-resistance plates containing ampicillin (Amp) and kanamycin (Kam) to screen the NCIMB 10586 strain carrying the pK18-BR plasmid.

[0018] S1.3. Screening by sucrose plate culture and PCR verification resulted in the engineered strain 10586ΔB::R;

[0019] S2. The method for knocking out the rsmA gene and replacing the mupR gene is as follows:

[0020] S2.1. Amplify the upstream and downstream homology arms of the rsmA gene (upstream and downstream primers are SEQ ID NO.11 / SEQ ID NO.12 and SEQ ID NO.15 / SEQ ID NO.16, respectively) and the tac promoter-mupR gene-mupO gene-terminator sequence (using primers SEQ ID NO.13 / SEQ ID NO.14), fuse the three together, and clone them into the plasmid pK18mobsacB to obtain the recombinant plasmid pK18-NR.

[0021] S2.2. Transform the recombinant plasmid pK18-NR into E. coli S17 and co-culture it with the 10586ΔB::R strain. Screen positive clones using LB double-antibody plates containing Amp and Kam.

[0022] S2.3. Screening by sucrose plate culture and PCR verification resulted in the engineered strain 10586ΔBA::R;

[0023] S3. The method for knocking out the rsmN gene and replacing the mupR-mupO gene is as follows:

[0024] S3.1. Amplify the upstream and downstream homology arms of the rsmN gene (upstream and downstream primers are SEQ ID NO. 17 / SEQ ID NO. 18 and SEQ ID NO. 21 / SEQ ID NO. 22, respectively) and the tac promoter-mupR gene-terminator sequence (using primers SEQ ID NO. 19 / SEQ ID NO. 20), fuse the three together, and clone into pK18mobsacB to obtain the recombinant plasmid pK18-NR.

[0025] S3.2. Transform the recombinant plasmid pK18-NR into E. coli S17 and co-culture it with the 10586ΔBA::R strain. Screen positive clones using LB double-antibody plates containing Amp and Kam.

[0026] S3.3. The engineered strain 10586ΔBAN::R was obtained by sucrose plate culture screening and PCR verification.

[0027] In a fifth aspect, the present invention provides a mupirocin-producing strain constructed by the method described above.

[0028] In a sixth aspect, the present invention provides a culture medium for culturing the mupirocin-producing strain, comprising the following components: glycerol, glucose, tryptone, corn steep liquor, sodium chloride, ammonium chloride, and ammonium sulfate.

[0029] As one embodiment of the present invention, the culture medium includes the following components: 50-100 g / L glycerol, 10-40 g / L glucose, 20-50 g / L tryptone, 3-10 g / L corn steep liquor, 3-5 g / L sodium chloride, 0.5-1 g / L ammonium chloride, 0.5-1 g / L ammonium sulfate, and a pH of 6.2-6.8.

[0030] In a seventh aspect, the present invention provides a method for producing mupirocin, comprising: aerobically culturing the mupirocin-producing strain in the culture medium and fermenting the mupirocin.

[0031] As an embodiment of the present invention, the fermentation temperature is 22-28° C. and the pH is 5.0-7.0.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1. The present invention studies the application of the rsmA gene in improving the mupirocin production of Pseudomonas fluorescens and discovers for the first time that rsmA is a negative regulatory gene, that is, the mupirocin production of Pseudomonas fluorescens can be increased by knocking out the rsmA gene, reducing the expression of the rsmA gene, or reducing the activity of the rsmA protein.

[0034] 2. The present invention is based on a metabolic regulation strategy, uses Pseudomonas fluorescens as a chassis strain, and integrates multiple copies of the mupR gene by knocking out the regulatory genes rsmA, rsmN and xylB genes, thereby providing a mupirocin genetically engineered high-yield strain 10586ΔBAN::R; wherein rsmA is a negative regulatory site, and rsmN and xylB are neutral sites, all of which are natural sites of the genome, are relatively stable, and are not easily lost during the passage process; at the same time, knocking out rsmA and replacing it with the mupR gene can not only utilize the expression of the mupR gene to increase the mupirocin yield, but also eliminate the negative regulatory effect of rsmA, thereby synergistically increasing the mupirocin yield.

[0035] 3. The mupirocin-producing genetically engineered strain 10586ΔBAN::R constructed by the present invention can produce up to 7,561 mg / L of mupirocin, including 7,060 mg / L of pseudomonic acid A, providing a solid foundation for the industrial production of mupirocin. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Other features, objects and advantages of the present invention will become more apparent upon reading the detailed description of non-limiting embodiments with reference to the following drawings:

[0037] Figure 1 The mupirocin yields of the genetically engineered strain 10586ΔrsmA and the wild-type strain in Example 1;

[0038] Figure 2 is the mupirocin yield of the genetically engineered strains 10586ΔxylB and 10586ΔrsmN and the wild-type strain in Example 2; wherein A is the mupirocin yield of the genetically engineered strain 10586ΔxylB and the wild-type strain, and B is the mupirocin yield of the genetically engineered strain 10586ΔrsmN and the wild-type strain;

[0039] Figure 3 This is the xylB gene knockout (replacement) map in Example 3;

[0040] Figure 4 The electrophoretic diagrams of the xylB gene and its upstream and downstream fragments amplified by PCR using internal and external primers in Pseudomonas fluorescens NCIMB 10586 and the genetically engineered strain 10586ΔB::R in which xylB is replaced with mupR in Example 3 are shown;

[0041] Figure 5 The electrophoretic diagrams of PCR amplification of the rsmA gene and its upstream and downstream fragments using internal and external primers in the genetically engineered bacteria 10586ΔB::R and 10586ΔBA::R in which rsmA was replaced with mupR in Example 4 are shown;

[0042] Figure 6The electrophoretogram of PCR amplification of rsmN gene and its upstream and downstream fragments by internal and external primers for the genetically engineered bacteria 10586ABAN::R and 10586ABAN::R with rsmN replaced by mupR in Example 5, respectively;

[0043] Figure 7 Mupirocin production and growth curves of the genetically engineered bacterial strain 10586ABAN::R at different temperatures in Example 6;

[0044] Figure 8 Mupirocin production and growth curves of the genetically engineered bacterial strain 10586ABAN::R at different pHs in Example 6. DETAILED DESCRIPTION

[0045] The present application will be described in detail below with reference to the accompanying drawings and specific examples. The following examples are implemented on the premise of the technical solutions of the present application, and provide detailed implementation modes and specific operation processes, which will help those skilled in the art to further understand the present application. It should be pointed out that the protection scope of the present application is not limited to the following examples, and several adjustments and improvements made on the premise of the concept of the present application all belong to the protection scope of the present application.

[0046] Example 1: Verification of rsmA as a negative regulatory gene

[0047] In this example, the rsmA gene in the Pseudomonas fluorescens NCIMB 10586 genome is knocked out, and a genetically engineered bacterial strain 10586ArsmA is constructed, and fermentation experiments are carried out to compare the yield with that of the wild type, to verify the influence of gene knockout on the yield, and the process is as follows:

[0048] 1) Using NCIMB 10586 genomic DNA as a template, appropriate primer pairs SEQ ID NO.11 / SEQ ID NO.12 and SEQ ID NO.15 / SEQ ID NO.16 were designed on either side of the gene to amplify approximately 500 bp of homology arms upstream and downstream, respectively, by PCR. The PCR product fragment size was verified by agarose gel electrophoresis, and the target DNA fragment was recovered. A fusion PCR method was used to obtain the target sequence approximately 500 bp upstream and downstream of rsmA. This target sequence was then ligated with linearized pK18mobsacB digested with EcoRI and HindIII restriction enzymes. The ligated product was heat-shock transformed into E. coli DH5α competent cells. Screening was performed using LB plates containing Kan, and single colonies were selected for colony PCR and plasmid sequencing verification. The correct recombinant plasmid pK18-rsmA was heat-shock transformed into E. coli S17. After activating the wild-type P. fluorescens NCIMB10586 strain twice on LB plates, single colonies were picked and transferred to 5 mL of LB medium. Cultured with shaking overnight at 28°C. 1.5 mL of fresh culture medium containing the wild-type P. fluorescens NCIMB 10586 strain and fresh culture medium containing the recombinant plasmid pK18-rsmA were each transferred to a sterile EP tube. Centrifuged at 6,000 rpm for 10 minutes, the supernatant discarded, and the remaining cells resuspended and rinsed in 1 mL of sterile LB liquid medium. Repeat this process twice. The obtained bacteria were resuspended in 100 μL LB and mixed, and then dropped onto an antibiotic-free LB plate and cultured at 28°C for 36-48 hours; the co-cultured bacteria were then transferred to an EP tube containing 200 μL LB, and the uniform mixed bacterial solution obtained after vortexing was spread on an LB double-antibody plate containing Amp and Kan, and cultured at 28°C for 36-48 hours; after colony PCR verification of the grown single colonies, the correct single colonies were resuspended in sterile ddH2O, and then spread on an LB plate containing 20% ​​sucrose, and cultured at 28°C for 48 hours; finally, the single colonies were photocopied onto an Amp LB plate and a Kan LB plate, respectively, and cultured at 28°C for 24 hours. The single colonies that grew on the Amp LB plate but not on the Kan LB plate were verified by bacterial PCR and sequenced. After colony PCR and sequencing verification, the genetically engineered strain 10586ΔrsmA was finally obtained.

[0049] 2) Mupirocin fermentation experiments were conducted in shake flasks using the same fermentation medium for P. fluorescens NCIMB 10586 wild-type strain and strain 10586ΔrsmA. The fermentation medium consisted of 50 g glycerol, 5 g glucose, 30 g tryptone, 5 g corn steep liquor, 5 g NaCl, 0.5 g NH4Cl, and 0.5 g (NH4)2SO4. The volume was made up to 1 L with ultrapure water, and the natural pH was approximately 6.5. For both P. fluorescens NCIMB 10586 wild-type strain and strain 10586ΔrsmA, the seed liquid was inoculated at 1% inoculum in 250 mL concave-angle flasks containing 60 mL of the corresponding liquid medium. The flasks were maintained at 28°C and 220 rpm under the same conditions.

[0050] Fermentation results such as Figure 1 As shown, the production of pseudomonic acid A of strain 10586ΔrsmA was significantly higher than that of the wild-type strain of P.fluorescens NCIMB 10586, indicating that knocking out the rsmA gene can increase the production of pseudomonic acid A, verifying that rsmA is a negative regulatory gene.

[0051] Example 2: Verification that xylB and rsmN are neutral sites

[0052] In this example, the xylB and rsmN genes were knocked out in the genome of Pseudomonas fluorescens NCIMB 10586, respectively, to construct genetically engineered strains 10586ΔxylB and 10586ΔrsmN. Fermentation experiments were conducted and the yields were compared with those of the wild type to verify the effect of the gene knockout on the yield. The process is as follows:

[0053] 1) Using genomic DNA from NCIMB 10586 as a template, appropriate primer pairs SEQ ID NO. 5 / SEQ ID NO. 6 and SEQ ID NO. 9 / SEQ ID NO. 10, as well as SEQ ID NO. 17 / SEQ ID NO. 18 and SEQ ID NO. 21 / SEQ ID NO. 22, were designed to amplify approximately 500 bp of upstream and downstream homology arms of the corresponding genes xylB and rsmN, respectively. The PCR product sizes were verified by agarose gel electrophoresis, and the target DNA fragments were recovered. Fusion PCR was used to obtain the upstream and downstream target sequences of xylB and rsmN, respectively. These sequences were then ligated with linearized pK18mobsacB digested with EcoRI and HindIII restriction enzymes. The ligation products were heat-shock transformed into E. coli DH5α competent cells. Screening was performed using LB plates containing Kan, and single colonies were selected for colony PCR and plasmid sequencing. The correct recombinant plasmid pK18-xylB was heat-shock transformed into E. coli S17 cells. After activating the wild-type P. fluorescens NCIMB 10586 strain twice on LB plates, single colonies were picked and transferred to 5 mL of LB medium and cultured with shaking at 28°C overnight. Simultaneously, E. coli S17 cells containing the recombinant plasmids pK18-xylB and pK18-rsmN were inoculated into 5 mL of LB medium containing Kan and cultured with shaking at 37°C overnight. 1.5 mL of each fresh culture medium containing the wild-type P. fluorescens NCIMB 10586 strain and the recombinant plasmids pK18-xylB and pK18-rsmN from E. coli S17 were transferred to sterile EP tubes and centrifuged at 6,000 rpm for 10 minutes. The supernatant was removed and the remaining cells were resuspended and rinsed in 1 mL of sterile LB liquid medium. This was repeated twice.The obtained E. coli S17 bacteria containing the recombinant plasmids pK18-xylB and pK18-rsmN were resuspended with the wild-type strain in 100 μL LB, placed for 1 hour, and then dropped onto an antibiotic-free LB plate and cultured at 28°C for 36-48 hours; the co-cultured bacteria were then transferred to an EP tube containing 200 μL LB, and the uniform mixed bacterial solution obtained after vortexing was spread on an LB double-antibody plate containing Amp and Kan, and cultured at 28°C for 36-48 hours; the grown single colonies were verified by colony PCR, and the correct single colonies were resuspended in sterile ddH2O, and then spread on an LB plate containing 20% ​​sucrose, and cultured at 28°C for 48 hours; finally, the single colonies were copied onto the Amp LB plate and the Kan LB plate, and cultured at 28°C for 24 hours. Single colonies grown on LB plates were verified by bacterial PCR and sequenced. After colony PCR and sequencing verification, the genetically engineered strains 10586ΔxylB and 10586ΔrsmN were finally obtained.

[0054] 2) Mupirocin fermentation experiments were conducted in shake flasks using the same fermentation medium for the genetically engineered strains 10586ΔxylB and 10586ΔrsmN. The fermentation medium consisted of 50 g glycerol, 5 g glucose, 30 g tryptone, 5 g corn steep liquor, 5 g NaCl, 0.5 g NH₄Cl, and 0.5 g (NH₄)₂SO₄, made up to 1 L with ultrapure water, with a natural pH of approximately 6.5. The seed liquid of the genetically engineered strains 10586ΔxylB and 10586ΔrsmN was inoculated with 1% of the seed liquid in 250 mL concave-angle flasks containing 60 mL of the corresponding liquid medium. The shake flasks were maintained at 28°C and 220 rpm under the same conditions.

[0055] Fermentation results such as Figure 2 As shown, the production of pseudomonic acid A of strains 10586ΔxylB and 10586ΔrsmN was close to that of the wild-type strain P.fluorescens NCIMB 10586, indicating that knocking out the xylB gene and rsmN gene had little effect on the production of pseudomonic acid A, verifying that both xylB and rsmN were neutral sites.

[0056] Example 3: Construction of engineered strain 10586ΔB::R

[0057] In this example, the xylB gene was replaced with the mupR gene in the genome of Pseudomonas fluorescens NCIMB 10586 to construct the genetically engineered strain 10586ΔB::R. The process is as follows:

[0058] 1) Using NCIMB 10586 genomic DNA as a template, suitable primer pairs SEQ ID NO.5 / SEQ ID NO.6 and SEQ ID NO.9 / SEQ ID NO.10 were designed on both sides of the gene to amplify the homology arms of about 500bp upstream and downstream respectively by PCR. Using the tac-mupR-terminator gene sequence as a template (located on the plasmid), a primer pair SEQ ID NO.7 / SEQ ID NO.8 was designed to amplify the tac promoter-mupR gene-terminator sequence. The DNA target fragment was recovered after verifying the fragment size of the PCR product by agarose gel electrophoresis. The fusion PCR method was used to obtain the xylB upstream-tac promoter-mupR gene-terminator-xylB downstream target sequence, which was enzyme-ligated with the linearized pK18mobsacB digested with EcoRI and HindIII restriction enzymes. The gene knockout (replacement) map is shown as follows: Figure 3 As shown, the enzyme-linked product was heat-shock transformed into E. coli DH5α competent cells. Screening was performed using LB plates containing Kan, and single colonies were selected for colony PCR and plasmid sequencing verification. The correct recombinant plasmid pK18-BR was heat-shock transformed into E. coli S17.

[0059] 2) Conjugation Transfer: First, after activating the wild-type P. fluorescens NCIMB 10586 strain twice on LB plates, a single colony was picked and transferred to 5 mL of LB medium. The culture was shaken overnight at 28°C. Simultaneously, E. coli S17 containing the recombinant plasmid pK18-BR was inoculated into 5 mL of LB medium containing Kan and shaken overnight at 37°C. 1.5 mL of each of these bacterial cultures was transferred to a sterile EP tube and centrifuged at 6,000 rpm for 10 min. The supernatant was removed and the remaining cells were resuspended and rinsed in 1 mL of sterile LB liquid medium. This was repeated twice. The obtained bacteria were resuspended in 100 μL LB and placed for 1 hour, then dropped onto an antibiotic-free LB plate and cultured at 28°C for 36-48 hours; the co-cultured bacteria were then transferred to an EP tube containing 200 μL LB, and the uniform mixed bacterial solution obtained after vortexing was spread on an LB double-antibody plate containing Amp and Kan, and cultured at 28°C for 36-48 hours; after colony PCR verification of the grown single colonies, the correct single colonies were resuspended in sterile ddH2O, and then spread on an LB plate containing 20% ​​sucrose, and cultured at 28°C for 48 hours; finally, the single colonies were photocopied onto an Amp LB plate and a Kan LB plate, respectively, and cultured at 28°C for 24 hours. The single colonies that grew on the Amp LB plate but not on the KanLB plate were verified by bacterial PCR and sequencing. After colony PCR and sequencing verification, the genetically engineered strain 10586ΔB::R was finally obtained.

[0060] Figure 4Electrophoresis diagrams of the xylB gene and its upstream and downstream fragments amplified by PCR using internal and external primers in Pseudomonas fluorescens NCIMB 10586 and the genetically engineered strain 10586ΔB::R, where xylB is replaced by mupR. M: Marker; Figure 4 Lane A 8-9: single exchange occurred; Figure 4 Lane B 1: Double crossover occurred, replacing the xylB gene locus. Amplification results showed that the xylB gene was successfully replaced by mupR.

[0061] Example 4: Construction of engineered strain 10586ΔBA::R

[0062] In this example, the rsmA gene was replaced with the mupR gene in the genome of the engineered strain 10586ΔB::R. The process for constructing the genetically engineered strain 10586ΔBA::R is as follows:

[0063] 1) Using NCIMB 10586 genomic DNA as a template, appropriate primer pairs SEQ ID NO.11 / SEQ ID NO.12 and SEQ ID NO.15 / SEQ ID NO.16 were designed to flank the gene and amplify approximately 500 bp of upstream and downstream homology arms, respectively, by PCR. Using the pSEVR plasmid as a template, primer pair SEQ ID NO.13 / SEQ ID NO.14 was designed to amplify the tac promoter-mupR gene-terminator sequence. The PCR product size was verified by agarose gel electrophoresis, and the target DNA fragment was recovered. A fusion PCR method was used to obtain the target sequence: rsmA upstream-tac promoter-mupR gene-terminator-rsmA downstream. This target sequence was then ligated with linearized pK18mobsacB digested with EcoRI and HindIII restriction enzymes. The ligation product was heat-shock transformed into E. coli DH5α competent cells. Screening was performed using LB plates containing Kan, and single clones were selected for colony PCR and plasmid sequencing verification. The correct recombinant plasmid pK18-AR was heat-shock transformed into E. coli S17.

[0064] 2) Take 1.5 mL of fresh culture liquid containing genetically engineered strain 10586AB::R and 1.5 mL of fresh culture liquid containing E. coli S17 containing recombinant plasmid pK18-AR into a sterile EP tube, centrifuge at 6,000 rpm for 10 min, remove the supernatant, and resuspend the remaining bacterial cells with 1 mL of sterile LB liquid medium and repeat 2 times. After resuspending the obtained bacterial cells with 100 μL of LB for 1 h, drop them on an antibiotic-free LB plate and incubate at 28°C for 36-48 h; then transfer the co-cultured bacterial cells to an EP tube containing 200 μL of LB, vortex to obtain a uniform mixed bacterial liquid, and then spread it on an LB double-antibiotic plate containing Amp and Kan, and incubate at 28°C for 36-48 h; after colony PCR verification of the grown single colonies, resuspend the correct single colonies in sterile ddH2O, and then spread them on an LB plate containing 20% sucrose and incubate at 28°C for 48 h; finally, copy the single colonies on an Amp LB plate and a Kan LB plate, and incubate at 28°C for 24 h, and perform bacterial cell PCR verification and sequencing comparison on the single colonies that grow on the Amp LB plate but not on the Kan LB plate, and finally obtain the genetically engineered strain 10586ABAN::R after colony PCR and sequencing verification.

[0065] Figure 5 The electrophoretograms of PCR amplification of rsmA gene and its upstream and downstream fragments by internal and external primers for genetically engineered bacteria 10586AB::R and 10586ABAN::R in which rsmA is replaced by mupR. Among them, M: Marker; Figure 5 Lane 3, 6: single exchange occurs; Figure 5 Lane 7: double exchange occurs, replacing the rsmA gene site. The amplification results show that the rsmA gene has been successfully replaced by mupR.

[0066] Example 5: Construction of genetically engineered strain 10586ABAN::R

[0067] In this example, the rsmN gene is replaced by the mupR gene on the genome of the genetically engineered strain 10586ABAN::R, and the process of constructing the genetically engineered strain 10586ABAN::R is as follows:

[0068] 1) Using NCIMB 10586 genomic DNA as a template, appropriate primer pairs SEQ ID NO. 17 / SEQ ID NO. 18 and SEQ ID NO. 21 / SEQ ID NO. 22 were designed to flank the gene and amplify approximately 500 bp of upstream and downstream homology arms, respectively, by PCR. Using the pSEVR plasmid as a template, primer pair SEQ ID NO. 19 / SEQ ID NO. 20 was designed to amplify the tac promoter-mupR gene-terminator sequence. The PCR product size was verified by agarose gel electrophoresis, and the target DNA fragment was recovered. A fusion PCR method was used to obtain the target sequence (rsmN upstream-tac promoter-mupR gene-terminator-rsmN downstream). This sequence was then ligated with linearized pK18mobsacB digested with EcoRI and HindIII restriction enzymes. The ligation product was heat-shock transformed into E. coli DH5α competent cells. Screening was performed using LB plates containing Kan, and single colonies were selected for colony PCR and plasmid sequencing verification. The correct recombinant plasmid pK18-NR was heat-shock transformed into E. coli S17.

[0069] 2) Transfer 1.5 mL of fresh culture medium containing the genetically engineered strain 10586ΔBA::R and 1.5 mL of fresh culture medium of E. coli S17 containing the recombinant plasmid pK18-NR to sterile EP tubes. Centrifuge at 6,000 rpm for 10 min. Remove the supernatant and resuspend the remaining cells in 1 mL of sterile LB liquid medium. Repeat this process twice. The obtained bacteria were resuspended in 100 μL LB and mixed for 1 hour, then dropped onto an antibiotic-free LB plate and cultured at 28°C for 36-48 hours; the co-cultured bacteria were then transferred to an EP tube containing 200 μL LB, and the uniform mixed bacterial solution obtained after vortexing was spread on an LB double-antibody plate containing Amp and Kan, and cultured at 28°C for 36-48 hours; after colony PCR verification of the grown single colonies, the correct single colonies were resuspended in sterile ddH2O, and then spread on an LB plate containing 20% ​​sucrose, and cultured at 28°C for 48 hours; finally, the single colonies were photocopied onto an Amp LB plate and a Kan LB plate, respectively, and cultured at 28°C for 24 hours. The single colonies that grew on the Amp LB plate but not on the Kan LB plate were verified by bacterial PCR and sequenced. After colony PCR and sequencing verification, the genetically engineered strain 10586ΔBAN::R was finally obtained.

[0070] Figure 6 Electrophoresis diagrams of the rsmN gene and its upstream and downstream fragments amplified by PCR using internal and external primers in the genetically engineered strain 10586ΔBA::R and 10586ΔBAN::R, where rsmN was replaced by mupR. M: Marker; Figure 6 Lane A 1-3: single exchange occurred; Figure 6 Lanes B, 3 and 4: Double crossover occurred, replacing the rsmN locus. Amplification results indicate that the rsmN gene has been successfully replaced by mupR.

[0071] The specific primer sequences are shown in Table 1

[0072] Table 1 Primer sequences

[0073]

[0074]

[0075] Example 6: Temperature and pH conditions for the production of mupirocin using 10586ΔBAN::R

[0076] This example relates to fermentation conditions for mupirocin production.

[0077] 1) Mupirocin fermentation medium (1 L): 85 g glycerol, 30 g glucose, 30 g tryptone, 5 g corn steep liquor, 5 g NaCl, 0.5 g NH4Cl, 0.5 g (NH4)2SO4, and make up with ultrapure water to a natural pH of approximately 6.5.

[0078] 2) Temperature Gradient Single-factor Experiment: For the temperature gradient experiment, the 10586ΔBAN::R strain was inoculated with 1% seed solution in a 250 mL concave-angle flask containing 60 mL of the corresponding liquid medium. The shake flasks were then incubated at 20, 22, 24, 26, 28, 30, and 32°C at a rotation speed of 220 rpm, and fermentation was performed under the same conditions.

[0079] 3) The seed solution was inoculated at 1% in a 5-L fermentor containing 3 L of MP-2 medium. The culture temperatures were set at 22, 24, and 26°C, respectively. The pH was maintained at 5.0, 5.5, 6.0, 6.5, and 7.0 by automated feed of 8 M H2SO4 (30%) and 10 M NaOH. The culture was shaken at 24°C and 220 rpm. Samples were taken every 12 hours to determine the relative concentrations of PA-A and PA-B until their concentrations stabilized.

[0080] Figure 7 The mupirocin production of the genetically engineered strain 10586ΔBAN::R at different temperatures ( Figure 7 A) and growth curve ( Figure 7 B).

[0081] Figure 8 The mupirocin production of the genetically engineered strain 10586ΔBAN::R at different pH values ​​( Figure 8 A) and growth curve ( Figure 8 B).

[0082] High-concentration mupirocin production was achieved by fermentation of the genetically engineered strain 10586ΔBAN::R in mupirocin liquid culture medium. At 24°C and natural pH, the yields of pseudomonic acid A and B in 10586ΔBAN::R were 6,439 mg / L and 615 mg / L, respectively, for a total mupirocin yield of 7,054 mg / L. At 24°C and pH 6.0, the yields of pseudomonic acid A and B were 7,060 mg / L and 501 mg / L, respectively, for a total mupirocin yield of 7,561 mg / L.

[0083] Mupirocin was fermented by the genetically engineered strain 10586ΔB::R and the genetically engineered strain 10586ΔBA::R in mupirocin liquid medium 2 at 24°C, pH 6.0, and a rotation speed of 220 rpm. The yields of mupirocin and pseudomonic acid A of the engineered strain 10586ΔB::R were 2,619 and 3,436 mg / L, respectively, and the yields of mupirocin and pseudomonic acid A of the engineered strain 10586ΔBA::R were 5,050 and 5,832 mg / L, respectively.

[0084] By fermenting mupirocin in mupirocin liquid medium 2 at 24°C, pH 6.0, and a rotation speed of 220 rpm, the genetically engineered strain 10586ΔBAN::R achieved high mupirocin yields. The pseudomonic acid A yield of 10586ΔBAN::R was 7,060 mg / L, the pseudomonic acid B yield was 501 mg / L, and the total mupirocin yield was 7,561 mg / L.

[0085] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various variations or modifications within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. The use of the rsmA gene in increasing the mupirocin production of Pseudomonas fluorescens, characterized in that: The nucleotide sequence of the rsmA gene is shown in SEQ ID NO.1; The method for increasing the mupirocin production of Pseudomonas fluorescens comprises one or more of knocking out the rsmA gene, reducing the expression of the rsmA gene, and reducing the activity of the rsmA protein.

2. The use according to claim 1, characterized in that The Pseudomonas fluorescens includes the P. fluorescens NCIMB 10586 strain.

3. Use of an inhibitor of the rsmA gene in increasing the mupirocin production of Pseudomonas fluorescens, characterized in that: The inhibitor includes one or more of an agent for knocking out the rsmA gene, an agent for reducing the expression of the rsmA gene, and an agent for reducing the activity of the rsmA protein.

4. A method for constructing a strain for producing mupirocin, characterized in that: Pseudomonas fluorescens is used as a base strain, and the mupirocin-producing strain is obtained by knocking out the rsmA gene and replacing it with the mupR gene.

5. A method for constructing a strain for producing mupirocin, characterized in that: Pseudomonas fluorescens is used as a base strain, and the mupirocin-producing strain is obtained by knocking out the rsmA, rsmN and xylB genes and integrating the mupR genes at corresponding positions.

6. The construction method according to claim 4 or 5, characterized in that: The nucleotide sequence of the rsmN gene is shown in SEQ ID NO.2; the nucleotide sequence of the xylB gene is shown in SEQ ID NO.3; and the nucleotide sequence of the mupR gene is shown in SEQ ID NO.

4.

7. A mupirocin-producing strain constructed according to the method of any one of claims 4 to 6.

8. A culture medium for culturing the mupirocin-producing strain according to claim 7, characterized in that: The culture medium comprises the following components: 50-100 g / L glycerol, 10-40 g / L glucose, 20-50 g / L tryptone, 3-10 g / L corn steep liquor, 3-5 g / L sodium chloride, 0.5-1 g / L ammonium chloride, 0.5-1 g / L ammonium sulfate, and has a pH of 6.2-6.

8.

9. A method for producing mupirocin, characterized in that: The method comprises: aerobically culturing the mupirocin-producing strain according to claim 7 in the culture medium according to claim 8, and fermenting the mupirocin.

10. The method according to claim 9, characterized in that The fermentation temperature is 22-28° C., and the pH is 5.0-7.0.

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