A method and application of increasing lincomycin production by modifying the gene of Streptomyces lincomycin SLCG_2951

Abstract

The present invention provides a method for increasing the yield of lincomycin by modifying the SLCG_2951 gene of Streptomyces lincomycin, knocking out the SLCG_2951 gene of Streptomyces lincomycin by genetic engineering, obtaining a high-yield strain of lincomycin, and fermenting and producing lincomycin A with the obtained strain; wherein the nucleotide sequence of the SLCG_2951 gene is shown in SEQ ID NO.1. The present invention also provides an application of the above method. The present invention screens the SLCG_2951 gene that affects the mycelium morphology, knocks out the SLCG_2951 gene on the genome of Streptomyces lincomycin by genetic engineering, and can delay the mycelium breakage of Streptomyces lincomycin A134 on a fermentation tank, thereby increasing the yield of lincomycin A, and providing technical support for increasing the yield of lincomycin in industrial production.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to a method and application of increasing the yield of lincomycin by transforming the gene of Streptomyces lincomycin SLCG_2951. Background Art

[0002] Streptomyces has evolved a complex morphological differentiation cycle in nature. First, spores grow in a suitable environment to form germinated spores. Germinated spores continue to grow at the top to form branched multinucleate vegetative hyphae. Under the action of hydrophobic proteins, the vegetative hyphae break the surface tension between the culture medium and the air to grow into aerial hyphae. Aerial hyphae then form diaphragms, which then grow to form uninucleate spore chains. Finally, the spore chains break to form individual spores. This morphological differentiation cycle is conducive to the survival and reproduction of Streptomyces in nature, and many studies have shown that the synthesis of active products is closely related to the complex and diverse morphological differentiation of Streptomyces.

[0003] The morphology of Streptomyces is closely related to the yield of antibiotics, and different antibiotics have different requirements for the morphology of mycelium, so Streptomyces will form significantly different mycelium morphologies during liquid fermentation. Ideally, the mycelium is loose, which makes it easier for cells to absorb nutrients. However, the actual situation is not the case. For some Streptomyces, loose mycelium can increase the yield, but for other Streptomyces, mycelium balls are more conducive to increasing the yield. For example, the loose mycelium of Streptomyces coelicolor is prone to form broken mycelium, which will increase the yield of prodigiosin, but reduce the yield of actinomycin. When the mycelium of Streptomyces avermitilis is balled, it is more conducive to increasing the yield of avermectin, and when the mycelium of Streptomyces freundii is balled, it is also conducive to increasing the yield of tylosin. When the mycelium of Streptomyces noursii, which is a loose mycelium in the wild type, is balled, it is conducive to increasing the yield of nystatin. The size of the mycelium ball of Saccharopolyspora erythrorhizium will also affect the yield of erythromycin. When the mycelium ball is large, the yield of erythromycin will increase, while when the mycelium ball is small, its yield will decrease. When Streptomyces hygroscopicus formed small mycelial balls, the production of geldenin increased significantly. However, the production of clavulanic acid and virginiamycin produced by Streptomyces clavuliformis and Streptomyces virginica did not change significantly, whether in mycelial balls or loose mycelium.

[0004] Lincomycin is a lincosamide antibiotic, produced industrially by Streptomyces lincomycin. Due to its excellent performance in tissue and cell penetration, it can be used clinically to treat various infectious diseases caused by Gram-positive bacteria, such as acute tracheitis and pneumonia. At the same time, the new drug clindamycin synthesized from its active ingredient lincomycin A has a more significant antibacterial effect, and its derivative products such as clindamycin metronidazole liniment can be used to remove acne and repair skin, etc., and have good market prospects. Therefore, scholars today attach great importance to improving the fermentation yield of lincomycin.

[0005] In view of the relationship between the aforementioned Streptomyces morphology and antibiotic production, the present invention attempts to screen and transform the mycelium morphology-related genes of Streptomyces lincomycin, and to increase the lincomycin production by regulating the mycelium morphology expression of the strain. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a method and application for increasing the yield of lincomycin by transforming the gene of Streptomyces lincomycin SLCG_2951.

[0007] The present invention adopts the following technical solutions to solve the above technical problems:

[0008] A method for improving the yield of lincomycin by modifying the lincomycin Streptomyces lincomycin SLCG_2951 gene, knocking out the lincomycin Streptomyces lincomycin SLCG_2951 gene through genetic engineering to obtain a lincomycin high-yield strain, and using the obtained strain to ferment and produce lincomycin A; wherein the nucleotide sequence of the SLCG_2951 gene is shown in SEQ ID NO.1.

[0009] As one of the preferred embodiments of the present invention, the amino acid sequence encoded by the SLCG_2951 gene is shown in SEQ ID NO.2.

[0010] As one of the preferred embodiments of the present invention, the deletion of the SLCG_2951 gene can delay the mycelium breakage of the corresponding Streptomyces lincomycin in the fermentation tank and increase the yield of lincomycin A.

[0011] As one of the preferred embodiments of the present invention, the lincomycin Streptomyces is specifically lincomycin Streptomyces A134, which is a modified strain obtained by mutation on the basis of the existing lincomycin engineered bacteria; the lincomycin Streptomyces A134 was sent to the China General Microbiological Culture Collection Center at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing on May 6, 2024 for preservation, and it was found to be alive. Its classification name is Streptomyces lincolnensis, and its preservation number in the China General Microbiological Culture Collection Center (CGMCC) is CGMCC: 30506; the lincomycin Streptomyces A134 can produce high levels of lincomycin A (the active ingredient of lincomycin) during the fermentation process, but almost no lincomycin B, effectively reducing the downstream separation and purification costs, and meeting more economical production needs.

[0012] An application of the method for increasing the yield of lincomycin by modifying the SLCG_2951 gene of Streptomyces lincomycin: knocking out the SLCG_2951 gene in an industrial strain of Streptomyces lincomycin to obtain a high-yield mutant for lincomycin A production.

[0013] Design ideas and principles:

[0014] The advantage of Streptomyces lincomycin A134 is that it can produce high lincomycin A (the active ingredient of lincomycin) during the fermentation process, but almost no lincomycin B. However, compared with other high-yield Streptomyces lincomycin, the overall lincomycin production of Streptomyces lincomycin A134 is slightly insufficient. The present invention observes and compares the differences between it and other Streptomyces lincomycin throughout the production process, and finds that the mycelium of Streptomyces lincomycin A134 breaks relatively early, and based on this, it is speculated whether "delaying the mycelium breakage of Streptomyces lincomycin A134 in the fermentation tank can increase the yield of lincomycin".

[0015] Next, the present invention studied and analyzed the genes of Streptomyces lincomycin A134, and finally screened out a SLCG_2951 gene sequence that can affect the mycelium morphology, and verified through genetic engineering that the SLCG_2951 gene can delay the mycelium breakage of Streptomyces lincomycin A134 in the fermentation tank, thereby increasing the yield of lincomycin A.

[0016] The advantages of the present invention compared to the prior art are:

[0017] In the study of the present invention, the SLCG_2951 gene that affects the mycelium morphology was screened. By knocking out the SLCG_2951 gene on the genome of Streptomyces lincomycin through genetic engineering, the mycelium breakage of Streptomyces lincomycin A134 on the fermentation tank can be delayed, thereby increasing the yield of lincomycin A, providing technical support for increasing the yield of lincomycin in industrial production.

[0018] After knocking out SLCG_2951 in Streptomyces lincomycin A134, in a 15L fermenter, A134ΔSLCG_2951 delayed mild mycelial breakage to 52h, and severe mycelial breakage to 96h, which was 16h later than that of the unmodified A134. At the same time, the lincomycin A production of A134ΔSLCG_2951 reached 4.42g / L, which was 41.6% higher than that of A134, and its bacterial concentration during the fermentation process was always better than that of A134.

[0019] In summary, the deletion of the SLCG_2951 gene of the present invention can effectively delay the mycelium breakage of the lincomycin A strain A134 in the fermentation tank, restore the mycelial growth, and thus increase the lincomycin A yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the location information map of the SACE_2951 gene and its neighboring genes on the genome;

[0021] Figure 2 is a schematic diagram of the construction of the ΔSLCG_2951 mutant;

[0022] Figure 3is the PCR identification diagram of ΔSLCG_2951 (in the figure, M: 5000 bp DNA Marker; +: pKC1139-Δ2951; -: A134; 1ΔSLCG_2951);

[0023] Figure 4 It is a result diagram of relevant parameters of the horizontal fermentation process of A134 and A134ΔSLCG_2951 fermenters (in the figure, Figure A is a curve diagram of bacterial concentration change in A134 and A134ΔSLCG_2951 fermenters; Figure B is a curve diagram of lincomycin A production change in A134 and A134ΔSLCG_2951 fermenters; Figure C is a curve diagram of reducing sugar change in A134 and A134ΔSLCG_2951 fermenters; Figure D is a curve diagram of amino nitrogen production change in A134 and A134ΔSLCG_2951 fermenters);

[0024] Figure 5 are the mycelium morphology observation pictures of A134 and A134ΔSLCG_2951 fermenters (in the figure, picture A is the horizontal mycelium morphology picture of A134 fermenter; picture B is the horizontal mycelium morphology picture of A134ΔSLCG_2951 fermentation);

[0025] Figure 6 These are scanning electron micrographs of the bacterial morphology of A134 and A134ΔSLCG_2951 after 24 hours (in the figure, Figure A is a scanning electron micrograph of the bacterial morphology of A134; Figure B is a scanning electron micrograph of the bacterial morphology of A134ΔSLCG_2951). DETAILED DESCRIPTION

[0026] The following is a detailed description of an embodiment of the present invention. This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiment.

[0027] The strains and plasmids used in the following examples are shown in Table 1, and the synthetic primer sequences are shown in Table 2.

[0028] The E. coli used in the following examples were cultured in liquid LB medium at 37°C or on solid LB plates supplemented with 1.25% agar. The lincomycin-producing bacteria Streptomyces lincomycin were cultured in tryptone soy broth (TSBY) medium at 30°C or on modified Gould's 1 (MGM) plates containing 1.8% agar.

[0029] Lysozyme, TES, PEG3350, and apramycin used in the following examples were purchased from Sigma. TSB, casamino acids, yeast extract, and peptone were purchased from Oxoid. Glycine, agar, and other chemicals were purchased from a reagent company. Routine operations of Escherichia coli and Streptomyces lincomycin were performed according to standard operating techniques. DNA synthesis and sequencing were commissioned to Sangon Biotech (Shanghai) Co., Ltd.

[0030] Table 1 The present invention relates to strains and plasmids

[0031]

[0032]

[0033] Table 2 The present invention relates to primers

[0034]

[0035] Example 1

[0036] SLCG_2951 gene related information:

[0037] Based on the gene annotation information of KEGG database (https: / / www.kegg.jp / ), the location information of SLCG_2951 and its surrounding genes in the genome of Streptomyces lincolnii and their potential gene functions are as follows Figure 1 shown.

[0038] The nucleotide sequence of the SLCG_2951 gene is shown in SEQ ID NO.1, with a total length of 3357 bp; the encoded amino acid sequence is shown in SEQ ID NO.2, consisting of 1118 amino acids.

[0039] Example 2

[0040] Construction of SLCG_2951 knockout strain (see Figure 2 ):

[0041] Using the A134 genome as the amplification template, 2951-P1 / P2 and 2951-P3 / P4 were used as primers to amplify 1.5 kb of the upstream and downstream homology arms of SLCG_2951, respectively, and recovered using a gel recovery kit. Then, the upstream and downstream homology arms were digested with XbaI / EcoRI and XbaI / HindIII, respectively, and the digested fragments were gel-recovered and the DNA was quantified using a UV spectrophotometer. At the same time, the pKC1139 plasmid was double-digested with EcoRI / HindIII and then recovered and quantified. According to the quantitative results, the digested plasmid, upstream homology arm, and downstream homology arm were mixed in a ratio of 1:10:10, T4 ligase was added, and the water bath connection process was carried out at 22°C. Then, all the ligation products were transformed into Escherichia coli DH5α, and then coated on an LB plate containing 0.1% Apr and cultured for 16 hours to grow a single clone. Finally, a single clone was picked and cultured in liquid culture medium for expansion and plasmid extraction to obtain a DH5α-positive strain carrying plasmid pKC1139-Δ2951.

[0042] The obtained pKC1139-Δ2951 plasmid was transformed into the protoplasts of A134 via pEG3350 and homologous recombination technology ( Figure 2 ) Complete the construction of SLCG_2951 knockout strain, the specific experimental operation is as follows:

[0043] Take about 4000-5000ng of pKC1139-Δ2951 and mix it with A134 protoplasts, then add pEG3350, spread the mixture on an R5 plate and culture it at 30℃ for 20h. When membranous bacteria grow on its surface, add Apr antibiotics for covering and screening. After 3-5 days, the single clone that grows on the plate and has a black bottom is the single exchange strain, which is enriched on an industrial plate containing Apr and cultured at 30℃ for 3-5 days. Subsequently, scrape the enriched spores, dilute and spread them on an antibiotic-free R5 plate, and culture at a constant temperature of 37℃ for 5-6 days to lose the plasmid. Finally, spread half of the single clones on the R5 plate on an Apr industrial plate and half on an antibiotic-free industrial plate. Select the single clone that does not grow on Apr but grows on the antibiotic-free plate for enrichment. After it produces spores, scrape a certain amount of spores as a template, and use 2951-P5 and 2951-P6 as primers for PCR identification. Figure 3 , and obtain the correct knockout strain.

[0044] Example 3

[0045] Detection of lincomycin production in A134 and A134ΔSLCG_2951 fermenters (15 L):

[0046] The fermentation performance of A134 and A134ΔSLCG_2951 was tested in a fermenter (15 L).

[0047] The results are as follows Figure 4 As shown: After the deletion of SLCG_2951, by observing the bacterial concentration change curve ( Figure 4 A) It was found that the overall bacterial concentration of A134ΔSLCG_2951 during the fermentation period was significantly better than that of A134. The bacterial growth of A134ΔSLCG_2951 during the entire fermentation period was significantly more stable, and also improved to a certain extent in the later stage of fermentation.

[0048] The lincomycin A production curve ( Figure 4 B) It was found that the yield of A134ΔSLCG_2951 was basically the same as that of A134 in the early stage of fermentation. After 144 hours of fermentation, the yield of A134ΔSLCG_2951 was always higher than that of A134, and the maximum yield could reach 4.42g / L, which was 41.6% higher than that of A134 (3.12g / L).

[0049] By observing the glucose consumption curve ( Figure 4 C) found that A134ΔSLCG_2951 had a stronger absorption capacity for carbon sources during the fermentation cycle; the amino nitrogen content curve ( Figure 4 D) There is no significant difference in nitrogen source utilization between the two.

[0050] Example 4

[0051] Observation of mycelial morphology in A134 and A134ΔSLCG_2951 fermentation tanks (15L):

[0052] Based on the fermentation performance test of A134 and A134ΔSLCG_2951 in the fermentation tank (15L) in Example 3, this example combined the mycelium morphology of A134 and A134ΔSLCG_2951 for observation.

[0053] The results are as follows Figure 5 As shown: A134ΔSLCG_2951 did not produce mycelium breakage at 36h of fermentation, slight breakage occurred at 52h, and a large number of mycelium breakage occurred at 96h ( Figure 5 A and 5B).

[0054] The above results showed that the absence of SLCG_2951 delayed the mycelial breakage of Streptomyces lincomycin A134, increased the concentration of mycelials during the fermentation cycle, and thus increased the yield of lincomycin A. The mycelial morphology and yield analysis of A134 and A134ΔSLCG_2951 fermenters showed that SLCG_2951 affected the yield of lincomycin by participating in the mycelial breakage of Streptomyces lincomycin A134.

[0055] Example 5

[0056] Scanning electron microscopy observation of mycelium of A134 and A134ΔSLCG_2951:

[0057] The difference in the fine structure of the mycelium of A134 and A134ΔSLCG_2951 can be further observed by scanning electron microscopy. First, take 1 mL of the fermentation broth sample of A134 and A134ΔSLCG_2951 24h fermenter, centrifuge at 4000rpm for 10min; wash the bacterial precipitate with PBS buffer until the supernatant is clear, and the culture medium is basically washed; then, add 1 mL of 2.5% glutaraldehyde to the washed bacterial precipitate, and fix the bacteria overnight at 4℃. After fixation, wash the fixative with PBS buffer, prefreeze for 12h, dehydrate in a freeze vacuum dryer for 12h, and finally steam gold in a gold steamer 3 times, 30s each time, so that the conductive particles are attached to the surface of the sample, and then it can be observed on the machine.

[0058] The results are as follows Figure 6 Shown: Electron microscopic images of mycelium of A134 and A134ΔSLCG_2951 at 24h ( Figure 6 A, B) It can be seen that the end of the mycelium of A134 is as rough as the cross section of a cut rope, while the end of the mycelium of A134ΔSLCG_2951 is very smooth and is a complete mycelium; the fracture incision in the electron microscopy photo of the 36h mycelium of A134 is even more obvious, showing obvious signs of fracture.

[0059] The above results further proved the mycelium breakage phenomenon of A134.

[0060] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for modifying Streptomyces lincomyces SLCG_2951 A method for genetically increasing lincomycin production, characterized in that: Knockout of Streptomyces lincomyces by genetic engineering SLCG_2951 Gene, obtain lincomycin high-yield strain, and use the obtained strain to ferment and produce lincomycin A; wherein the lincomycin Streptomyces is specifically lincomycin Streptomyces A134, which is deposited in China General Microbiological Culture Collection Center with a deposit number of CGMCC: 30506; SLCG_2951 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The method according to claim 1, wherein the modified Streptomyces lincomyces SLCG_2951 A method for genetically increasing lincomycin production, characterized in that: Said SLCG_2951 The amino acid sequence encoded by the gene is shown in SEQ ID NO.

2.

3. The method according to claim 1 for modifying Streptomyces lincomyces SLCG_2951 A method for genetically increasing lincomycin production, characterized in that: Said SLCG_2951 The deletion of the gene can delay the breakage of the mycelium of the corresponding Streptomyces lincomycin in the fermentation tank and increase the production of lincomycin A.

4. A modified Streptomyces lincomyces as claimed in any one of claims 1 to 3 SLCG_2951 The application of the method for increasing lincomycin production by gene is characterized in that: Knockout in industrial strains of Streptomyces lincomyces SLCG_2951 gene, and obtained a high-yield mutant for lincomycin A production.

Images