Method and application for increasing lincomycin production by modifying Streptomyces lincolnensis SLCG_6736 gene

Abstract

The present invention relates to the field of genetic engineering technology and provides a method for increasing lincomycin production by modifying the SLCG_6736 gene of Streptomyces lincomycin. The SLCG_6736 gene is deleted in Streptomyces lincomycin through genetic engineering to obtain a high-yield engineered lincomycin strain, and the obtained strain is used to ferment and produce lincomycin. The nucleotide sequence of the SLCG_6736 gene is shown in SEQ ID NO. 1. The present invention also provides an application of the above method. The advantages of the present invention are that the negative regulator of lincomycin biosynthesis, SLCG_6736, was screened in the present invention's research. By knocking out the SLCG_6736 gene through genetic engineering, a high-yield lincomycin strain can be obtained, providing technical support for increasing lincomycin fermentation yield in industrial production.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to a method for increasing the yield of lincomycin by modifying the gene of Streptomyces lincomycin SLCG_6736 and its application. Background Art

[0002] Microorganisms can produce a rich variety of structurally diverse secondary metabolites, representing a treasure trove for natural medicine research and development. Actinomycetes are the primary source of clinically used microbial natural medicines, including half of all antimicrobial drugs. These substances possess antibacterial, antifungal, antitumor, immunosuppressive, and insecticidal activities, and are widely used in the pharmaceutical, agricultural, and food industries.

[0003] Streptomyces lincomycin is a key actinomycete in the industrial fermentation production of lincomycin. Its active component A is used to treat infections caused by Gram-positive bacteria such as Staphylococcus aureus and Streptococcus pneumoniae, as well as certain anaerobic Gram-negative bacteria. Lincomycin binds to the central loop of the 23S rRNA of the bacterial 50S ribosomal subunit, inhibiting the localization of tRNA within the peptidyl transferase center and thus affecting protein biosynthesis. Lincomycin has strong tissue and cell penetration and is widely used clinically.

[0004] With the maturity of genome sequencing, genome mining and gene editing technology of Streptomyces lincomycin, it was found that in addition to the currently known genes, the genome of Streptomyces lincomycin also contains a large number of unexpressed silent gene clusters. These gene clusters need to be screened to obtain the high-yield strains required for industrial production. In the past, industrial production strains were mainly obtained by physical or chemical mutagenesis methods, but traditional mutagenesis techniques have problems such as time-consuming and large randomness, and cannot provide theoretical guidance for breeding. The purpose of the present invention is to obtain lincomycin high-yield strains by directing gene changes through genetic engineering approaches for the production of lincomycin or intermediates.

[0005] NmrA (Nitrogen metabolite repression A) acts as a repressor of nitrogen metabolism, regulating the activity of the AreA transcription factor. Glutamine, a signaling molecule for intracellular nitrogen levels, is recognized by the NmrA protein. In an environment containing a preferred nitrogen source, NmrA is activated and binds to the AreA transcription factor, altering the conformation of the AreA protein, causing it to lose its ability to bind DNA and ultimately inhibiting the expression of genes involved in secondary nitrogen metabolism. In the absence of preferred nitrogen sources such as ammonium nitrogen and glutamine, NmrA dissociates from AreA, and active AreA binds to the promoter regions of genes involved in secondary nitrogen metabolism, thereby activating their transcription. Studies have found that when Streptomyces lincomycin is involved in antibiotic biosynthesis, the addition of a certain amount of nitrate affects the primary metabolism of the antibiotic and, in turn, affects the production of the antibiotic. Based on this, the present invention screened the SLCG_6736 gene, which is highly homologous to the nmrA gene, in Streptomyces lincomycin through bioinformatics comparison analysis, and attempted to transform the SLCG_6736 gene of Streptomyces lincomycin to increase the lincomycin production. 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 modifying the gene of Streptomyces lincomycin SLCG_6736.

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

[0008] A method for increasing lincomycin production by modifying the SLCG_6736 gene of Streptomyces lincomycin comprises deleting the SLCG_6736 gene in Streptomyces lincomycin through genetic engineering to obtain a high-yield engineered lincomycin strain, and using the obtained strain to ferment and produce lincomycin; wherein the nucleotide sequence of the SLCG_6736 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_6736 gene is shown in SEQ ID NO.2.

[0010] As one of the preferred embodiments of the present invention, the SLCG_6736 gene product negatively regulates lincomycin biosynthesis.

[0011] An application of the above method is to knock out the SLCG_6736 gene in an industrial strain to obtain a high-yield mutant strain that can be used for lincomycin production.

[0012] As one of the preferred embodiments of the present invention, the industrial strain is specifically selected as the high-yield strain LA219X.

[0013] The advantages of the present invention over the prior art are:

[0014] In the present invention, the negative regulator of lincomycin biosynthesis SLCG_6736 was screened out. By knocking out the SLCG_6736 gene through genetic engineering, a high-yield lincomycin strain was obtained, providing technical support for increasing the fermentation yield of lincomycin in industrial production.

[0015] Specifically, knocking out the SLCG_6736 gene in Streptomyces lincomycin LCGL increased lincomycin production by 16.6%, indicating that SLCG_6736 is a negative regulator of lincomycin biosynthesis. Using the high-yielding strain LA219X as a starting strain, knocking out the SLCG_6736 gene on its chromosome increased lincomycin production by 13.2%, demonstrating that the technique of knocking out the SLCG_6736 gene to increase lincomycin production is also applicable to industrial high-yielding strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the location information map of the SLCG_6736 gene and its neighboring genes on the chromosome;

[0017] Figure 2 is a schematic diagram of the construction of ΔSLCGL_6736;

[0018] Figure 3 is the PCR identification result of ΔSLCGL_6736 (in the figure, “M”: 5000 bp DNA Marker; “+”: pKC1139-Δ6736; “−”: LCGL; “1”: ΔSLCGL_6736);

[0019] Figure 4 PCR identification results of ΔSLCGL_6736 / pIB139-6736 complementation and LCGL / pIB139-6736 overexpression strains (in the figure, "M": 5000 bp DNA Marker; "+": pIB139 plasmid; "-": ultrapure water; "1": ΔSLCGL_6736 / pIB139-6736 complementation strain; "2": LCGL / pIB139-6736 overexpression strain; PCR product is Apr resistance gene, 776 bp);

[0020] Figure 5 This is the PCR identification result of the ΔSLA219X_6736 strain (in the figure, “M”: 5000 bp DNA Marker; “+”: pIB139 plasmid; “−”: ultrapure water; “1”: ΔSLA219X_6736);

[0021] Figure 6This is the analysis of lincomycin production of the starting strain LCGL and the ΔSLCGL_6736 strain (in the figure, “*”: P < 0.1);

[0022] Figure 7 The effect of the SLCG_6736 gene on strain morphological differentiation and the determination of the biomass of ΔSLCGL_6736 (Figure, Panel A shows the spore growth of LCGL and ΔSLCGL_6736 strains, where the left is LCGL strain and the right is ΔSLCGL_6736; Panel B shows the dry weight of mycelium of LCGL and ΔSLCGL_6736 strains);

[0023] Figure 8 The lincomycin production of LCGL, ΔSLCGL_6736, ΔSLCGL_6736 / pIB139-6736, and LCGL / pIB139-6736 strains was analyzed (in the figure, “*”: P<0.1, “ns”: no significant difference).

[0024] Figure 9 is the analysis of the transcription levels of genes related to the lincomycin biosynthesis gene cluster (in the figure, "*": P < 0.1, "**": P < 0.01, "***": P < 0.001, "ns": no significant difference);

[0025] Figure 10 This is the analysis of lincomycin production in the lincomycin high-producing strains LA219X and ΔSLA219X_6736 (in the figure, "***": P < 0.001). DETAILED DESCRIPTION

[0026] The following is a detailed description of an embodiment of the present invention. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process. However, 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 synthesized primer sequences are shown in Table 2. Among them, Streptomyces lincone LCGL is an improved strain obtained based on Streptomyces lincone LC-G (GenBank: CP022744.1), specifically: the SLCG_7011 gene (GenBank: AXG58166.1) of Streptomyces lincone LC-G is replaced with 4×attBΦC31; the 4×attBΦC31 is a 240 bp base sequence containing four attBΦC31 sites, the base sequence of which is shown in SEQ ID NO. 3.

[0028] Escherichia coli used in the following examples was cultured in liquid LB medium at 37°C or on solid LB medium supplemented with 1.25% agar. Streptomyces lincomycin was cultured in tryptone soy broth (TSBY) medium at 30°C or on modified Gouldian GM plates containing 1.8% agar.

[0029] PEG3350, lysozyme, TES, thiostrepton, and apramycin were purchased from Sigma. TSB, yeast extract, and peptone were purchased from Oxoid. Glycine, agar powder, sodium chloride, and other biological reagents were purchased from Sangon Biotech (Shanghai) Co., Ltd. General techniques for Escherichia coli and Streptomyces lincomycin were performed according to standard procedures. Primer synthesis and DNA sequencing were performed by Sangon Biotech (Shanghai) Co., Ltd.

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

[0031]

[0032]

[0033] Table 2 Primers of the present invention

[0034]

[0035]

[0036]

[0037] Example 1

[0038] SLCG_6736 gene related information:

[0039] The location of SLCG_6736 gene and its neighboring genes on chromosomes can be found in Figure 1 .

[0040] The specific nucleotide sequence of the SLCG_6736 gene is shown in SEQ ID NO.1, which is 861 bp in length. The encoded amino acid sequence is shown in SEQ ID NO.2, and the protein monomer size is approximately 29 KDa.

[0041] Example 2

[0042] Construction of SLCG_6736 gene deletion mutant (see Figure 2 ):

[0043] Using the LCGL genome as a template, primers 6736-P1, 6736-P2, 6736-P3, and 6736-P4 were used to amplify 1.5 kb of each upstream and downstream homology arms of SLCG_6736. The upstream and downstream homology arms were digested with HindIII / XbaI and KpnI / EcoRI, respectively, at 37°C for recovery and quantification. Simultaneously with the homology arms, the pKC1139 plasmid was double-digested with EcoRI / HindIII, recovered, and quantified. Based on the quantification results, the digested plasmid, upstream homology arms, and downstream homology arms were mixed at a ratio of 1:10:10 and ligated with T4 ligase at 22°C. The ligation product was then transformed into Escherichia coli DH5α and diluted and plated onto apramycin (Apr)-resistant LB plates for approximately 12 hours at 37°C to allow single colonies to grow. Finally, single clones were picked and transferred to LB liquid medium containing Apr resistance for expansion culture, and then transferred to 5 mL of LB medium containing Apr resistance and cultured for 12 hours to extract the pKC1139-Δ6736 plasmid.

[0044] The obtained pKC1139-Δ6736 plasmid was transformed into LCGL protoplasts via PEG3350, and the SLCG_6736 knockout strain was constructed using homologous large fragment recombination technology. The specific steps are as follows:

[0045] 3000 ng of pKC1139-Δ6736 was mixed with 50 μL of LCGL protoplasts, followed by the addition of 200 mL of PEG3350 and the mixture allowed to stand for 5 minutes. The mixture was diluted and spread onto R5 plates and incubated at 30°C for approximately 20 hours. Once membranous cells developed on the surface, Apr antibiotic was added for overlay screening. After 4 days, single colonies with black bottoms emerged from the plates. These colonies were enriched on industrial plates containing Apr and incubated at 30°C for 3 days. Subsequently, the enriched spores were selected, diluted, and spread onto antibiotic-free R5 plates. Incubated at 37°C for 3 days to eliminate the plasmid. Finally, a portion of the single colonies from the R5 plates were spread onto Apr industrial plates, and the remaining colonies were spread onto antibiotic-free industrial plates. Select the single clone that cannot grow on the Apr plate but can grow on the non-antibiotic plate and culture it until it produces spores. Then scrape a certain amount of spores into sterile water, boil them at high temperature for 15 minutes, and then perform PCR identification using 6736-P5 and 6736-P6 as primers. Figure 3 , and obtain the correct knockout strain.

[0046] Example 3

[0047] Construction of SLCG_6736 gene complementation and overexpression strains:

[0048] Using the LCGL genome as a template, primers 6736-P7 and 6736-P8 were used to amplify the SLCG_6736 gene fragment, which contained NdeI and XbaI restriction sites at both ends. The SLCG_6736 fragment and the pIB139 plasmid were then digested with NdeI and XbaI and recovered. The SLCG_6736 and pIB139 plasmids were ligated with T4 ligase at 21°C and transformed into Escherichia coli DH5α. The fragment was plated onto an Apr-resistant LB plate and incubated at 37°C for approximately 12 hours until a single colony emerged. The colony was then transferred to liquid LB medium and cultured for 5 hours before identification by PCR. Correctly identified cells were sent for sequencing and, if sequenced correctly, were preserved to obtain the pIB139-6736 plasmid.

[0049] The pIB139-6736 plasmid was introduced into LCGL and ΔSLCG_6736 protoplasts, respectively, by protoplast transformation. 20 h after transformation, the culture medium was covered with an aqueous solution of apramycin and cultured until transformants emerged. Transformants were selected and plated on industrial plates with Apr resistance. After the Apr-resistant transformants emerged, they were verified using primers Apr-F / R. Figure 4 As shown, the correct ΔSLCGL_6736 / pIB139-6736 and LCGL / pIB139-6736 strains were obtained.

[0050] Example 4

[0051] Construction of lincomycin high-producing strain ΔSLA219X_6736:

[0052] The pKC1139-Δ6736 plasmid was transformed into the protoplasts of LA219X via PEG3350 to construct the ΔSLA219X_6736 strain. The construction and screening method refer to Example 3, and the PCR identification results are as follows: Figure 5 .

[0053] Example 5

[0054] HPLC detection of lincomyces fermentation products:

[0055] After culturing Streptomyces lincomyces on the slant medium for 7 days, 1 cm 2 The spore mass was inoculated into a seed culture medium and cultured at 30°C, 240 rpm, and shaking for 48 hours. The culture was then transferred to a fermentation medium and cultured at 30°C, 240 rpm, and shaking for 7 days. Next, 2 mL of the bacterial culture was centrifuged at 12,000 rpm for 10 minutes. 200 μL of the supernatant was mixed with 800 μL of anhydrous ethanol and centrifuged at 12,000 rpm for 10 minutes. Finally, the supernatant was filtered through an organic filter membrane and transferred into a test bottle for yield analysis.

[0056] Example 6

[0057] Streptomyces lincomyces mycelium biomass detection:

[0058] LCGL and ΔSLCGL_6736 were inoculated with equal inoculum sizes into 5 mL of liquid TSBY and incubated at 30°C on a shaker for 48 h. Equal amounts of fresh LCGL and ΔSLCGL_6736 cultures were inoculated into YMG medium and incubated at 30°C and 240 rpm for 7 days. Every 24 hours, 1 mL of fresh culture was removed and stored in a -20°C refrigerator. After fermentation, the 7-day sample was centrifuged at 12,000 rpm for 10 minutes, and the supernatant discarded. The cells were washed with 1 mL of anhydrous ethanol and centrifuged at 12,000 rpm for 10 minutes, with the supernatant discarded. The wet cells were placed in a 65°C oven, dried for 2 days, and weighed. The cell mass was recorded. The dry weight of ΔSLCGL_6736 and LCGL cells was plotted against growth time to create a biomass curve.

[0059] Example 7

[0060] Transcriptional analysis of related genes in ΔSLCG_6736:

[0061] 24h LCGL and ΔSLCG_6736 bacterial cultures were collected and the required RNA was obtained using the Full-Form Gold RNA Extraction Kit. After conversion into cDNA, it was detected using a real-time fluorescence quantitative PCR instrument.

[0062] Example 8

[0063] Analysis of the results of the above embodiments:

[0064] 1. SLCG_6736 negatively regulates the biosynthesis of lincomycin

[0065] The SLCG_6736 gene deletion mutant has been identified and verified by PCR ( Figure 3 After 7 days of fermentation, the lincomycin production of ΔSLCGL_6736 was detected by HPLC. It was found that the production of lincomycin of ΔSLCGL_6736 was about 16.6% higher than that of LCGL. Figure 6 The above results confirmed that SLCG_6736 is a negative regulatory gene for lincomycin biosynthesis in Streptomyces lincomycin. Knocking out the SLCG_6736 gene in Streptomyces lincomycin by genetic engineering can increase lincomycin production.

[0066] 2. Effect of SLCG_6736 gene deletion on spore morphological differentiation and bacterial growth

[0067] To determine whether the SLCG_6736 gene regulates spore formation, LCGL and ΔSLCGL_6736 were simultaneously plated on MGM medium and cultured at 30°C for 7 days. The spore growth of the strains was observed daily. The results showed that there was no significant difference in the spore morphology of LCGL and ΔSLCGL_6736 ( Figure 7 A), indicating that the deletion of the SLCG_6736 gene does not affect spore formation. The dry weight of LCGL and ΔSLCGL_6736 was measured and the corresponding change curve was drawn. The results showed that the biomass of ΔSLCGL_6736 was not much different from that of LCGL ( Figure 7 B), suggesting that the deletion of the SLCG_6736 gene did not affect the primary metabolism of the bacteria.

[0068] 3. SLCG_6736 gene complementation and overexpression

[0069] To further verify that the increased lincomycin production in ΔSLCGL_6736 was due to the deletion of the SLCG_6736 gene, the SLCG_6736 gene expression vector pIB139-6736 was introduced into the protoplasts of LCGL and ΔSLCGL_6736, respectively. PCR identification yielded the complemented strain ΔSLCGL_6736 / pIB139-6736 and the overexpression strain LCGL / pIB139-6736. LCGL and ΔSLCGL_6736 strains were fermented in shake flasks. HPLC analysis showed that the lincomycin production in ΔSLCGL_6736 was 16.6% higher than that in LCGL; the lincomycin production in the complemented strain ΔSLCGL_6736 / pIB139-6736 was restored to the level of lincomycin in LCGL; and the lincomycin production in LCGL / pIB139-6736 was 16.2% lower than that in LCGL. Figure 8 These results indicate that SLCG_6736 negatively regulates the biosynthesis of lincomycin.

[0070] 4. Transcriptional analysis of related genes in ΔSLCG_6736

[0071] qRT-PCR data showed that compared with LCGL, the transcription levels of lmbA, lmbC, lmbD, lmbV, lmbZ, lmbJ, lmbK, lmrA, lmrB, lmrC, and lmbU in the lincomycin biosynthesis gene cluster in ΔSLCGL_6736 were upregulated to varying degrees ( Figure 9 ), indicating that SLCG_6736 negatively regulates the transcription level of genes in the cluster, thereby controlling the biosynthesis of lincomycin.

[0072] 5. Knockout of the SLCG_6736 gene in the high-yield strain LA219X can increase lincomycin production

[0073] The ΔSLA219X_6736 strain and the high-yield strain LA219X were activated by plating and then inoculated into shake flasks containing industrial seed culture medium. After 48 hours of incubation at 30°C and 240 rpm, the strains were transferred to fermentation medium and cultured for another 168 hours. After fermentation, the extracts were concentrated and analyzed by HPLC. Compared to LA219X, the lincomycin yield of ΔSLA219X_6736 increased by 13.2% ( Figure 10 ), which indicates that the SLCG_6736 gene in the high-yielding strain LA219X is also involved in controlling the production of lincomycin.

[0074] 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 scope of protection of the present invention.

Claims

1. A method for modifying Streptomyces lincomyces SLCG_6736 The method for genetically increasing lincomycin production is characterized in that: Through genetic engineering, SLCG_6736 Gene deletion is performed to obtain a strain with improved lincomycin production, and the obtained strain is used to produce lincomycin by fermentation; wherein, SLCG_6736 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. Application of a strain with improved lincomycin production, characterized in that: The strain with improved lincomycin production is obtained by the method according to claim 1; knocking out SLCG_6736 The gene is used to obtain the strain with improved lincomycin production, which is used for lincomycin production.

3. The use according to claim 2, characterized in that The industrial strain specifically selected is strain LA219X.

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