A method and application for improving lincomycin production by modifying the SLCG_1979 gene of Streptomyces lincomyces.

Abstract

This invention relates to the field of genetic engineering technology and provides a method for increasing lincomycin production by modifying the SLCG_1979 gene in *Streptomyces lincomyces*. The method involves deleting the SLCG_1979 gene, which encodes a TetR family transcription factor, from the *Streptomyces lincomyces* strain through genetic engineering, thereby obtaining a high-yielding lincomycin-producing strain. The resulting strain is then used for fermentation to produce lincomycin. The nucleotide sequence of the SLCG_1979 gene is shown in SEQ ID NO. 1. This invention also provides an application of the above-mentioned method for increasing lincomycin production in industrial strains. The advantage of this invention is that deleting the SLCG_1979 gene on the *Streptomyces lincomyces* chromosome through genetic engineering can yield a high-yielding lincomycin-producing strain, providing technical support for increasing lincomycin fermentation yield.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a method and application for increasing lincomycin production by modifying the SLCG_1979 gene of Streptomyces lincomyces. Background Technology

[0002] Actinomycetes, a group of Gram-positive bacteria that primarily reproduce by spores and grow in filamentous form, produce secondary metabolites such as streptomycin, kanamycin, spinosad, and lincomycin, accounting for two-thirds of clinically used antibiotics. In addition, actinomycetes are used as immunomodulators and receptor antagonists, making them indispensable to human life. Streptomyces is the largest genus in the order Actinomycetes, producing the most diverse range of antibiotics. Based on the sequencing and analysis of the entire genome of Streptomyces, it has been discovered that, in addition to currently known antibiotics, the Streptomyces genome contains a large number of gene clusters for the biosynthesis of secondary metabolites. However, these gene clusters are either silent or have weak biosynthetic capabilities, requiring screening to obtain high-yielding strains for industrial production. Previously, industrial production strains were mainly obtained through physical or chemical mutagenesis methods, but traditional mutagenesis techniques suffer from time-consuming and highly random problems, failing to provide theoretical guidance for breeding. The purpose of this invention is to obtain high-yielding lincomycin strains through genetic engineering to directionally modify genes for the production of lincomycin or intermediate products.

[0003] *Streptomyces lincomyces* is an important antibiotic-producing bacterium. The first lincomycin-producing strain, *Streptomyces lincomyces* NRRL2936, was obtained by Mason et al. in 1962 from soil near Nebraska, USA. Secondary metabolites produced during the fermentation process of *Streptomyces lincomyces* include lincomycin A and lincomycin B. Lincomycin A is the main active substance and is a widely used lincosamide antibiotic, clinically applied for infections caused by Gram-positive bacteria, particularly those caused by *Staphylococcus aureus* and *Micrococcus luteus*. It can also serve as a raw material for the chemical synthesis of clindamycin. By binding to the 23S rRNA central loop of the 50S subunit of sensitive ribosomes, it reduces the activity of peptidyl transferases, inhibiting peptide chain elongation and thus suppressing bacterial protein synthesis. Its antibacterial spectrum is similar to that of erythromycin.

[0004] Currently, research on the biosynthetic pathway of lincomycin is relatively mature, but the molecular regulatory mechanism of lincomycin biosynthesis is still being reported and improved.

[0005] TetR family regulatory factors are among the most common prokaryotic transcription regulators, widely distributed in bacteria and playing important roles in various physiological processes, including transcriptional regulation of antibiotic biosynthesis, multidrug efflux, responses to osmotic stress, and cell differentiation. With the development of structural biology, the spatial structures of TetR family proteins have been elucidated, mainly comprising an N-terminal DNA-binding domain and a C-terminal ligand-binding domain, exhibiting dual capabilities of DNA binding and ligand response. However, there are currently few reports on increasing lincomycin production in *Streptomyces lincosae* by altering regulatory factors. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a method and application for increasing lincomycin production by modifying the SLCG_1979 gene of Streptomyces lincomyces.

[0007] The present invention solves the above-mentioned technical problems by adopting the following technical solutions:

[0008] A method for increasing lincomycin production by modifying the SLCG_1979 gene of Streptomyces lincomyces involves deleting the SLCG_1979 gene encoding a TetR family transcription factor in Streptomyces lincomyces through genetic engineering to obtain a high-yielding strain of lincomycin. The obtained strain is then used for fermentation to produce lincomycin. The nucleotide sequence of the SLCG_1979 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_1979 gene is shown in SEQ ID NO.2.

[0010] As one of the preferred embodiments of the present invention, the SLCG_1979 gene expression product negatively regulates lincomycin biosynthesis.

[0011] The above-mentioned method for increasing lincomycin yield is applied to industrial strains by knocking out the SLCG_1979 gene in industrial strains to obtain high-yielding mutant strains for lincomycin production.

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

[0013] The advantages of this invention compared to the prior art are:

[0014] In this study, the negative regulator of lincomycin biosynthesis, SLCG_1979, was screened. By deleting the SLCG_1979 gene on the chromosome of Streptomyces lincomycin through genetic engineering, a high-yielding strain of lincomycin can be obtained, providing technical support for improving the fermentation yield of lincomycin.

[0015] Knocking out the SLCG_1979 gene in *Streptomyces lincomyces* LCGL increased lincomycin production by 27%, while reintroducing the SLCG_1979 gene into the ΔSLCGL_1979 deletion mutant restored lincomycin production, indicating that SLCG_1979 is a negative regulator of lincomycin biosynthesis. Using the high-yielding strain LA219X as the starting strain, knocking out the SLCG_1979 gene on its chromosome increased lincomycin production by 17.8%, demonstrating that the technique of knocking out SLCG_1979 to increase lincomycin production is also applicable to industrial strains. Attached Figure Description

[0016] Figure 1 This is a diagram showing the location of the SLCG_1979 gene and its neighboring genes on the chromosome.

[0017] Figure 2 This is a schematic diagram illustrating the construction of the ΔSLCGL_1979 mutant of this invention;

[0018] Figure 3 PCR identification of the ΔSLCGL_1979 mutant (in the figure, M: 5000bp DNA Marker; +: pKC1139-Δ1979 plasmid; -: LCGL; 1, 2: ΔSLCGL_1979 strain);

[0019] Figure 4 This is the PCR identification of strain ΔSLA219X_1979 (M: 5000bp DNA Marker; +: pKC1139-Δ1979 plasmid; lane 1: ΔSLA219X_1979 strain);

[0020] Figure 5 Analysis of lincomycin production in the starting strain LCGL, the deletion mutant ΔSLCGL 1979, the deletion-corrected strain and the corrected empty vector control strain, the overexpressing strain and the overexpressing empty vector control strain:

[0021] Figure 6 The effects of the SLCG_1979 gene on the morphological differentiation of the strain and the determination of the biomass of the ΔSLCGL_1979 mutant strain (Figure A shows the spore growth of the ΔSLCGL_1979 mutant strain and the original strain LCGL strain, where right: ΔSLCGL_1979 mutant strain, left: LCGL strain; Figure B shows the mycelial dry weight determination of the ΔSLCGL_1979 mutant strain and the original strain LCGL strain).

[0022] Figure 7This is an analysis of the transcriptional levels of genes related to the lincomycin biosynthesis gene cluster (in the figure, Figure A shows the transcriptional level analysis of regulatory genes; Figure B shows the transcriptional level analysis of resistance genes; and Figure C shows the transcriptional level analysis of structural genes).

[0023] Figure 8 Analysis of lincomycin yield in high-yield industrial lincomycin strains LA219X and ΔSLA219X_1979. Detailed Implementation

[0024] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operation processes. However, the scope of protection of the present invention is not limited to the following embodiments.

[0025] The strains and plasmids used in the following examples are shown in Table 1, and the synthesized primer sequences are shown in Table 2. Specifically, *Streptomyces lincosae* LCGL is an improved strain obtained based on *Streptomyces lincosae* LC-G (GenBank: CP022744.1), specifically by replacing the SLCG_7011 gene (GenBank: AXG58166.1) of *Streptomyces lincosae* LC-G with 4×attBΦC31; the 4×attBΦC31 is a 240bp sequence containing four attBΦC31 sites, as shown in SEQ ID NO. 3.

[0026] The *Escherichia 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. *Streptomyces lincomycin*, the lincomycin-producing bacterium, was cultured in tryptone soybean broth (TSBY) medium at 30°C or on modified Gauzes 1 (MGM) plates containing 1.8% agar.

[0027] PEG3350, lysozyme, TES, casein amino acids, thiosporin, and apramycin were purchased from Sigma-Aldrich. TSB, yeast extract, and peptone were purchased from Oxoid. Glycine, agar powder, sodium chloride, and other biological reagents were purchased from reagent companies. General handling techniques for *E. coli* and *Streptomyces lincosae* were performed according to standard operating procedures. Primer synthesis and DNA sequencing were performed by Sangon Biotech (Shanghai) Co., Ltd.

[0028] Table 1. This invention relates to strains and plasmids.

[0029]

[0030]

[0031] Table 2 This invention relates to primers.

[0032]

[0033]

[0034]

[0035] Example 1

[0036] Information related to the SLCG_1979 gene:

[0037] For the location of the SLCG_1979 gene and its neighboring genes on the chromosome, see [link to chromosome location information]. Figure 1 .

[0038] According to the LCGL genome information, the SLCG_1979 gene is 741 bp in length and the protein monomer size is 27.05 kDa.

[0039] Specifically, the specific nucleotide sequence of the SLCG_1979 gene is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2.

[0040] Example 2

[0041] Construction of the SLCG_1979 gene deletion mutant (see...) Figure 2 ):

[0042] Using LCGL genomic DNA as a template, primers 1979-P1, 1979-P2, 1979-P3, and 1979-P4 were used to amplify the upstream and downstream homologous arms of SLCG_1979, each 1.5 kb, and the amplified arms were recovered using a gel extraction kit. The upstream fragments of plasmids pUCTSR and SLCG_1979 were then digested, ligated, and transformed using restriction enzymes HindIII and XbaI. pUCTSR-1979-up was obtained by PCR and restriction enzyme digestion identification. The downstream fragments of pUCTSR-1979-up and SLCG_1979 were then digested, ligated, and transformed using restriction enzymes EcoRI and KpnI. pUCTSR-Δ1979 was obtained by PCR and restriction enzyme digestion identification. The pUCTSR-Δ1979 plasmid was then digested with restriction enzymes HindIII and EcoRI and cloned into the pKC1139 plasmid. The pKC1139-Δ1979 plasmid was obtained by bacterial PCR and restriction enzyme digestion verification.

[0043] The obtained pKC1139-Δ1979 plasmid was transformed into protoplasts: approximately 5000 ng of pKC1139-Δ1979 plasmid was added to 200 μL of PEG3350 and then added to the prepared *Streptomyces lincosae* LCGL protoplasts. After mixing, the mixture was allowed to stand for about 5 minutes. The protoplasts were then evenly spread on R5 medium and cultured for about 20 hours. When a thin bacterial film grew on the surface, 1 mL of sterile water and 20 μL of Apr antibiotic were added for screening. After culturing at 30°C for 4–5 days, single colonies with blackened bottoms emerged. These colonies were then enriched on industrial plates containing Apr and cultured at 30°C for 3–5 days. Spores were scraped and enriched, diluted, and spread onto antibiotic-free industrial plates. Incubation was carried out at 37°C for 2–3 days to allow plasmid relaxation and loss. Once single colonies appeared on the plates, half of the single colonies were placed on industrial plates containing TSR antibiotics, and the other half on industrial plates containing Apr antibiotics (50 μg / mL Apr and 30 μg / mL TSR). These were then incubated at 30°C for 3–4 days. Single colonies that did not grow on Apr but did grow on TSR plates were selected for enrichment. After sporulation, a certain amount of spores were scraped off as templates. PCR identification was performed using primers 1979-P5 and 1979-P6. The template for the positive control was the pKC1139-Δ1979 plasmid, and the template for the negative control was the LCGL genome. Verification was based on the size of the PCR product bands. Figure 3 The correct knockout strain was obtained.

[0044] Example 3

[0045] SLCG_1979 gene complementation and construction of overexpression strains:

[0046] Using LCGL as a template and primers 1979-P7 and 1979-P8, the SLCG_1979 gene with NdeI and XbaI restriction sites at both ends was amplified. The amplified and recovered SLCG_1979 fragment and pIB139 plasmid were double-digested with NdeI and XbaI. After recovery, the SLCG_1979 and pIB139 plasmids were ligated and transformed into *E. coli* DH5α. The cells were plated on LB agar plates containing Apr antibiotics and incubated at 37°C for approximately 16 hours, after which single colonies grew. Single colonies were selected and transferred to liquid LB medium for 5–8 hours of culture, followed by colony PCR identification. Correctly identified colonies were sequenced, and after successful sequencing, the pIB139-1979 plasmid was obtained through preservation.

[0047] The obtained pIB139-1979 plasmid was introduced into the protoplasts of the starting strains LCGL and ΔSLCGL_1979 via protoplast transformation. The empty pIB139 plasmid was also transformed into LCGL protoplasts using the same method. Approximately 24 hours after transformation, the culture medium was covered with apramycin aqueous solution and cultured until transformants appeared. Transformants were picked and plated on industrial plates containing Apr resistance. After Apr-resistant transformants appeared, PCR identification was performed using primers Apr-F / R to obtain the correct strains ΔSLCGL_1979 / pIB139, ΔSLCGL_1979 / pIB139-1979, LCGL / pIB139, and LCGL / pIB139-1979.

[0048] Example 4

[0049] Construction of the ΔSLA219X_1979 lincomycin-producing strain from the high-yield industrial strains:

[0050] The pKC1139-Δ1979 plasmid was transformed into LA219X protoplasts via PEG3350 to construct the ΔSLA219X_1979 strain. The construction and screening methods were as described above. PCR identification results are as follows. Figure 4 .

[0051] Example 5

[0052] HPLC detection of Streptomyces lincosum fermentation products:

[0053] After culturing *Streptomyces lincosum* on an agar slant for 7 days, a 1cm section was removed... 2 Inoculated into seed culture medium and cultured at 30℃ and 240 rpm for 48 h with shaking, then transferred to fermentation culture medium and cultured at 30℃ and 240 rpm for 7 days with shaking. Then, 2 mL of bacterial culture was centrifuged at 12000 rpm for 10 min. 200 μL of supernatant was then mixed with 800 μL of ethanol and centrifuged at 12000 rpm for 10 min. Finally, the supernatant was filtered through an organic filter membrane and injected into a test bottle for yield detection.

[0054] Example 6

[0055] Detection of Streptomyces lincosum mycelial biomass:

[0056] ΔSLCGL_1979 and LCGL were inoculated into 30 mL of liquid TSBY at the same inoculum size and cultured on a shaker at 30 °C for 48 h. Then, equal volumes of fresh LCGL and ΔSLCGL_1979 culture were transferred to YMG medium and cultured at 30 °C and 240 rpm for 7 days. During this period, 1 mL of culture was collected every 24 h and stored at -80 °C. After fermentation was complete, the seven-day sample was centrifuged at 12000 rpm for 10 min, and the supernatant was discarded. The cells were washed with 1 mL of anhydrous ethanol, centrifuged at 12000 rpm for 10 min, and the supernatant was discarded. The moistened cells were placed in a 65 °C oven for 2 days and weighed, and the cell mass was recorded. A biomass curve was plotted based on the dry weight of ΔSLCGL_1979 and LCGL cells according to the growth time.

[0057] Example 7

[0058] Transcriptional analysis of related genes in ΔSLCG_1979:

[0059] ΔSLCGL_1979 and LCGL bacterial cultures were collected after 24 hours. The required RNA was obtained using a full-length gold RNA extraction kit, reversed into cDNA, and then detected using a real-time quantitative PCR instrument.

[0060] Example 8

[0061] This embodiment analyzes the results of one of the above embodiments:

[0062] 1. The deletion mutant ΔSLCGL_1979 showed increased lincomycin production compared to the original strain LCGL.

[0063] The SLCG_1979 gene deletion mutant has been confirmed by PCR identification. Figure 3 After 7 days of fermentation in the fermentation medium, lincomycin production was determined by HPLC using ΔSLCGL_1979. The results showed that ΔSLCGL_1979 produced 27% more lincomycin than the original strain LCGL (see [link to original text]). Figure 5 HPLC results showed that SLCG_1979 is a negative regulator involved in lincomycin biosynthesis.

[0064] 2. SLCG_1979 gene reversion and overexpression

[0065] To verify that the increased lincomycin production in the mutant ΔSLCGL_1979 was due to the deletion of the SLCG_1979 gene, the SLCG_1979 gene expression vectors pIB139-1979 and pIB139 (as controls) were introduced into protoplasts of the ΔSLCGL_1979 mutant and the originating strain LCGL, respectively, to obtain reverting strains and empty vectors ΔSLCGL_1979 / pIB139-1979 and ΔSLCGL_1979 / pIB139. Subsequently, LCGL and the ΔSLCGL_1979 series of mutants were subjected to shake-flask fermentation. HPLC results showed that:

[0066] The lincomycin yield of ΔSLCGL_1979 was 27% higher than that of LCGL; the lincomycin yield of the recovering strain ΔSLCGL_1979 / pIB139-1979 was basically restored compared to LCGL; the lincomycin yield of LCGL / pIB139-1979 was lower than that of LCGL (see...). Figure 5 HPLC results further indicate that the SLCG_1979 gene can negatively regulate the biosynthesis of lincomycin.

[0067] 3. Effects of SLCG_1979 gene deletion on spore morphology differentiation and cell growth

[0068] To determine whether the SLCG_1979 gene regulates spore formation, the mutant strain ΔSLCGL_1979 and the control strain LCGL were simultaneously plated on slant agar plates and incubated at 30°C for 48h, 72h, 96h, and 120h to observe spore growth.

[0069] The results showed that the spore morphology of the ΔSLCGL_1979 mutant was significantly different from that of the LCGL mutant. Figure 6 A) indicates that the deletion of the SLCG_1979 gene affects spore formation.

[0070] The cell dry weights of the ΔSLCGL_1979 mutant and the LCGL strain were measured after 7 days of fermentation, and corresponding change curves were plotted. The results showed that the biomass of ΔSLCGL_1979 and LCGL was not significantly different (see...). Figure 6 B) suggests that the deletion of the SLCG_1979 gene did not affect the primary metabolism of the bacteria.

[0071] 4. SLCG_1979 negatively regulates the biosynthesis of lincomycin.

[0072] After culturing the ΔSLCGL_1979 mutant strain and its related strains in fermentation medium for 7 days, lincomycin production was analyzed by HPLC. The results showed that the ΔSLCGL_1979 mutant strain exhibited a significantly higher lincomycin production compared to the original strain. Figure 5qRT-PCR data showed that, compared with the original strain LCGL, the transcription levels of regulatory genes, resistance genes, and most structural genes within the lincomycin biosynthesis gene cluster in the ΔSLCGL_1979 mutant strain were increased to varying degrees. Figure 7 This indicates that SLCG_1979 negatively regulates the transcriptional level of genes within the cluster, thereby controlling the biosynthesis of lincomycin.

[0073] 5. The industrial strain ΔSLA219X_1979, which lacks the SLCG_1979 gene, can increase the yield of lincomycin.

[0074] The ΔSLA219X_1979 strain and the industrial strain LA219X were plated and activated, then inoculated into shake flasks containing industrial seed culture medium. After 48 h of incubation at 30℃ and 240 rpm, they were transferred to fermentation medium and cultured for another 168 h. After fermentation, extraction and concentration were performed, and HPLC analysis showed that the lincomycin yield of ΔSLA219X_1979 was increased by 17.8% compared to the starting strain LA219X. Figure 8 This indicates that the SLCG_1979 gene in the high-yielding strain LA219X is also involved in regulating lincomycin production.

[0075] 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 within the protection scope of the present invention.

Claims

1. A method for modifying Streptomyces lincosum SLCG_1979 A method for increasing lincomycin production through gene therapy, characterized in that, By using genetic engineering, the transcription factors encoding the TetR family in Streptomyces lincosalis LA219X were induced. SLCG_1979 Gene deletion was used to obtain a high-yield strain of lincomycin, and the obtained strain was used for fermentation to produce lincomycin; among which, SLCG_1979 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The method of modifying Streptomyces lincosum according to claim 1 SLCG_1979 A method for increasing lincomycin production through gene therapy, characterized in that, The SLCG_1979 The amino acid sequence encoded by the gene is shown in SEQ ID NO.

2.

3. The method of modifying Streptomyces lincosum according to claim 1 SLCG_1979 A method for increasing lincomycin production through gene therapy, characterized in that, The SLCG_1979 Gene expression products negatively regulate lincomycin biosynthesis.

4. A kind SLCG_1979 The application of genetically modified Streptomyces lincosinate is characterized by, The method constructed by any one of claims 1 to 3 SLCG_1979 Genetically modified Streptomyces lincomycin is used for the fermentation production of lincomycin.

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