Method for improving lincomycin yield by modifying streptomyces lincolnensis slcg_3615 gene and application thereof

Abstract

This invention provides a method for modifying Streptomyces lincosinate. SLCG_3615 A method to increase lincomycin production through gene therapy involves knocking out the TetR family transcriptional regulatory genes in Streptomyces lincomyces via genetic engineering. SLCG_3615 A high-yield engineered strain of lincomycin was obtained, and the obtained strain was used to produce lincomycin through fermentation; among which, SLCG_3615 The nucleotide sequence of the gene is shown in SEQ ID NO.1. This invention also provides... SLCG_3615 Applications of gene knockout modified *Streptomyces lincomycin*. In this study, the negative regulator of lincomycin biosynthesis, SLCG_3615, was screened and knocked out on the chromosome of *Streptomyces lincomycin* via genetic engineering. SLCG_3615 SLCG_3615 Gene copying can yield high-yield strains of lincomycin, providing technical support for increasing the fermentation yield of lincomycin in industrial production.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and more particularly to a method for modifying Streptomyces lincospores. SLCG_3615 Methods and applications of gene-based enhancement of lincomycin production. Background Technology

[0002] Streptomyces, as important producers of secondary metabolites, can synthesize a variety of bioactive secondary metabolites, including antifungal, antiviral, antitumor, and antihypertensive products. Antibiotics and immunosuppressants are the most important product types. With the rapid development of whole-genome sequencing technology, a large number of Streptomyces genome sequences have been analyzed. Studies have revealed that many of their biosynthetic gene clusters are often silent, or their ability to synthesize secondary metabolites is weak, making it difficult to meet the demands of industrial production for high-yield target products. Traditional methods for breeding high-yielding strains mostly rely on physical or chemical mutagenesis techniques. These methods are not only time-consuming, but also produce random and uncertain results, failing to provide clear theoretical guidance for strain breeding. Therefore, there is an urgent need to develop targeted and efficient strain modification technologies.

[0003] Lincomycin is a lincosamide antibiotic synthesized by *Streptomyces lincomycetes*, and its derivative, clindamycin, is also a core clinical antibiotic. Both have a well-defined mechanism of action: they act on the ribosomal peptidyl transferase center of Gram-positive bacteria and some Gram-negative bacteria, inhibiting peptide chain elongation by binding to the central loop of the 23S rRNA in the 50S subunit, thereby inhibiting bacterial protein synthesis and ultimately inhibiting bacterial growth and reproduction. In clinical applications, lincomycin is commonly used to treat bone and joint infections and can also effectively prevent postoperative intramural infections, oral infections, and skin and mucous membrane infections. It possesses advantages such as high efficacy, low toxicity, and low likelihood of cross-resistance, making it a promising antibiotic. Increasing its fermentation yield is of great significance for ensuring clinical drug availability.

[0004] TetR family transcriptional regulators (TFRs) are among the most widely distributed transcriptional regulators in prokaryotes, with actinomycetes exhibiting the highest number of TFRs and highly diverse regulatory mechanisms. These regulators act as "metabolic switches" and "precision regulators" within the cell, sensing intracellular and extracellular signaling molecules and precisely regulating the expression of numerous genes, including the gene cluster for antibiotic synthesis, a secondary metabolite in actinomycetes. This, in turn, regulates key cellular life activities such as primary and secondary metabolism. Based on the regulatory characteristics of TetR family regulators, targeted modification of related regulatory genes holds promise for achieving precise regulation of lincomycin biosynthesis, providing new insights for constructing high-yield lincomycin strains. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for modifying Streptomyces lincosum. SLCG_3615 Methods and applications of genes (TetR family genes) to increase lincomycin production.

[0006] The present invention solves the above-mentioned technical problems by adopting the following technical solutions:

[0007] A method of modifying Streptomyces lincospores SLCG_3615 A method to increase lincomycin production through gene therapy involves knocking out the TetR family transcriptional regulatory genes in *Streptomyces lincomyces* via genetic engineering. SLCG_3615 A high-yield engineered strain of lincomycin was obtained, and the obtained strain was used to produce lincomycin through fermentation; among which, SLCG_3615 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0008] As one of the preferred embodiments of the present invention, the target of the gene engineering approach for knockout is Streptomyces lincosum LA219X. SLCG_3615 Gene.

[0009] As one of the preferred embodiments of the present invention, the SLCG_3615 The amino acid sequence encoded by the gene is shown in SEQ ID NO.2.

[0010] As one of the preferred embodiments of the present invention, the SLCG_3615 Gene products negatively regulate lincomycin biosynthesis.

[0011] A sort of SLCG_3615 The application of gene knockout modified Streptomyces lincosinate, the aforementioned SLCG_3615 Gene knockout modified Streptomyces lincomyces was constructed using the above method and used for the fermentation production of lincomycin.

[0012] The advantages of this invention compared to the prior art are:

[0013] In this study, the negative regulator of lincomycin biosynthesis, SLCG_3615, was screened and knocked out on the chromosome of Streptomyces lincomycin via genetic engineering. SLCG_3615 Gene copying can yield high-yield strains of lincomycin, providing technical support for increasing the fermentation yield of lincomycin in industrial production.

[0014] Specifically, knockout in *Streptomyces lincosae* LCGL SLCG_3615 The gene was detected, resulting in a 21.8% increase in lincomycin production, indicating that SLCG_3615 is a negative regulator of lincomycin biosynthesis. Further investigation was conducted using the high-yielding strain LA219X as the starting strain, and the gene was knocked out on its chromosome. SLCG_3615 The gene can increase lincomycin production by 13.4%, indicating that knocking out the gene... SLCG_3615The techniques for increasing lincomycin yield are also applicable to high-yield industrial strains. Attached Figure Description

[0015] Figure 1 yes SLCG_3615 A diagram showing the location of a gene and its neighboring genes on a chromosome;

[0016] Figure 2 It is Δ SLCGL_3615 Construct a schematic diagram;

[0017] Figure 3 It is Δ SLCGL_3615 The PCR identification results are shown in the figure (in the figure, M: 5000bp DNA Marker; +: pKC1139-Δ3615; -: LCGL; 1: Δ SLCGL_3615 );

[0018] Figure 4 It is LA219XΔ 3615 PCR identification results of the strain (in the figure, M: 5000bp DNA Marker; +: pKC1139-3615 plasmid; -: LA219X; lane 1: LA219XΔ) 3615 strains);

[0019] Figure 5 The originating strains are LCGL and Δ SLCGL_3615 Analysis of lincomycin production (in the figure, Δ) SLCGL_3615- 1 Δ SLCGL_3615-2 Δ SLCGL_3615-3 For Δ SLCGL_3615 Three clones obtained from strain culture and screening were used for technical replication; "**": P < 0.01);

[0020] Figure ⑥ yes SLCG_3615 The influence of genes on strain morphological differentiation and Δ SLCGL_3615 Biomass measurement (in the figure, Figure A shows LCGL and Δ) SLCGL_3615 Spore growth of the strain, where, left: Δ SLCGL_3615 strain ， Right: LCGL strain; Figure B shows LCGL and Δ SLCGL_3615 (Determination of dry weight of mycelium of the strain).

[0021] Figure 7 This is a 24-hour transcriptional analysis of genes related to the lincomycin biosynthesis gene cluster (as shown in the figure: *). P <0.1, "**": P < 0.01, "***": P <0.001, “ns”: no significant difference);

[0022] Figure 8 It is a high-yield lincomycin strain LA219X, LA219XΔ 3615 Results of lincomycin production (in the figure, "**") P < 0.01). Detailed Implementation

[0023] 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.

[0024] 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 lincosae* LCGL is a strain improved from *Streptomyces lincosae* LC-G (GenBank: CP022744.1), specifically: the *Streptomyces lincosae* LC-G... SLCG_7011 The gene (GenBank:AXG58166.1) was replaced with 4×attBΦC31; the 4×attBΦC31 is a 240bp base sequence containing 4 attBΦC31 sites, and the base sequence is shown in SEQ ID NO. 3.

[0025] 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 lincosae* were cultured in tryptone soybean broth (TSBY) medium at 30°C or on modified Gauzes 1 (MGM) plates containing 1.8% agar.

[0026] PEG3350, lysozyme, TES, thiosphingolipids, and apramycin used in the following examples 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 *Escherichia coli* and *Streptomyces lincosae* were performed according to standard operating procedures. Primer synthesis and DNA sequencing were performed by Sangon Biotech (Shanghai) Co., Ltd.

[0027] Table 1. This invention relates to strains and plasmids.

[0028]

[0029] Table 2 This invention relates to primers.

[0030]

[0031] Example 1

[0032] SLCG_3615 Gene-related information:

[0033] SLCG_3615 For the location of the gene and its neighboring genes on the chromosome, see [link to relevant documentation]. Figure 1 .

[0034] According to LCGL genome information, SLCG_3615 The gene is 747 bp in length and the protein monomer is approximately 27.1 kDa in size.

[0035] Specifically, SLCG_3615 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2.

[0036] Example 2

[0037] SLCG_3615 Construction of gene deletion mutants (see) Figure 2 ):

[0038] Using the LCGL genome as a template, 3615-P1 、 3615-P2 、 3615-P3 、 Primers 3615-P4 were used to amplify the following results: SLCG_3615 Each of the upstream and downstream homologous arms, 1.5 kb in length, was recovered. At 37°C, [the following processes were performed] separately. Xba I / Hind III. Xb aI / Eco RI was used to digest and recover the upstream and downstream homologous arms, and then quantify them. Simultaneously, the pKC1139 plasmid was digested. Eco RI / Hind III. The plasmid was recovered and quantified after double digestion with enzymes III. Based on the quantification results, the digested plasmid, upstream homologous arm, and downstream homologous arm were mixed at a ratio of 1:10:10 and ligated with T4 ligase at 22°C. The ligation product was then transformed into *E. coli* DH5α and, at 37°C, diluted and plated on Apr-resistant LB agar plates. Single colonies grew after approximately 12 hours. Finally, single colonies were selected and expanded into Apr-resistant LB liquid medium, then transferred to 5 mL of Apr-resistant LB medium and cultured for 12 hours. The pKC1139-Δ3615 plasmid was then extracted.

[0039] The obtained pKC1139-Δ3615 plasmid was transformed into LCGL protoplasts via PEG3350-mediated transformation, and homologous large fragment recombination technology was used to complete the transformation. SLCG_3615Construction of the knockout strain. The specific experimental procedure is as follows: Approximately 6000 ng of pKC1139-Δ3615 was mixed with 50 μL of LCGL protoplasts, then 200 mL of PEG3350 was added. The mixture was allowed to stand for 5-10 min, then diluted and spread onto R5 plates. Incubation was carried out at 30°C for approximately 20 h until a membrane-like bacterial cell appeared on the surface. Apr antibiotic was then added for screening. After approximately 4 days, single colonies with blackened bases grew on the plates. These colonies were enriched on industrial plates containing Apr and incubated at 30°C for approximately 3 days. Subsequently, the enriched spores were picked, diluted, and spread onto antibiotic-free R5 plates, and incubated at 37°C for 3 days to induce plasmid loss. Finally, a portion of the monoclonal colonies from the R5 plate were plated onto Apr industrial plates, and the remaining monoclonal colonies were plated onto antibiotic-free industrial plates. Monoclonal colonies that could not grow on Apr plates but could grow on antibiotic-free plates were selected and cultured until spores were produced. A certain amount of spores were then scraped into sterile water and boiled at high temperature for 10 minutes. PCR identification was then performed using primers 3615-P5 and 3615-P6. Figure 3 The correct knockout strain Δ was obtained. SLCGL_3615 .

[0040] Example 3

[0041] Lincomycin high-yielding strain LA219XΔ 3615 Construction:

[0042] The pKC1139-Δ3615 plasmid was transformed into the protoplasts of LA219X via PEG3350-mediated transformation to construct the LA219XΔ 3615 The strain was constructed and screened using the method described in Example 2. PCR identification results are as follows: [[ID= .

[0043] Example 4

[0044] HPLC detection of Streptomyces lincosum fermentation products:

[0045] After culturing *Streptomyces lincosum* on slant agar for 7 days, 1 cm of... 2 Spore blocks were inoculated into seed culture medium and cultured at 30℃ and 240 rpm for 48 h with shaking. Then, they were transferred to fermentation 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 anhydrous 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.

[0046] Example 5

[0047] Detection of Streptomyces lincosum mycelial biomass:

[0048] LCGL and Δ were inoculated at the same amount.​ Inoculate into 5 mL of liquid TSBY, incubate at 30°C on a shaker for 48 h, then take equal amounts of LCGL and Δ ​ Fresh bacterial culture was cultured in YMG medium at 30℃ and 240 rpm for 7 days. During this period, 1 mL of fresh bacterial culture was taken every 24 hours and stored at -20℃. After fermentation was complete, the 7-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℃ oven for 2 days and weighed, and the cell mass was recorded. Δ ​ The cell dry weight of LCGL was used to plot the cell biomass curve based on the growth time.

[0049] Example 6

[0050] Δ ​ Transcriptional analysis of related genes in the middle:

[0051] Collect LCGL and Δ over 24 hours ​ The bacterial culture was used to obtain the required RNA using a full-gold RNA extraction kit. After being reverse-engineered into cDNA, it was detected using a real-time quantitative PCR instrument.

[0052] Example 7

[0053] Analysis of the results of one of the above embodiments in this example:

[0054] 1. SLCG_3615 negatively regulates the biosynthesis of lincomycin.

[0055] Δ ​ After fermentation in the fermentation medium for 168 hours, the lincomycin yield was detected by HPLC, and Δ was found to be... ​ Production increased by up to 21.8% compared to LCGL. ​ This has been confirmed. ​ It is a negative regulatory gene for lincomycin biosynthesis in *Streptomyces lincosae*, and its inactivation in *Streptomyces lincosae* is achieved through genetic engineering. ​ Genes can increase lincomycin production.

[0056] 2. Missing ​ The Influence of Genes on Spore Morphology Differentiation and Cell Growth

[0057] In order to determine ​ Whether the gene regulates the spore formation of the bacterial cell, and whether LCGL and Δ ​ Simultaneously, the strain was coated onto industrial plates and incubated at 30°C for 7 days, with daily observation of spore growth. Results showed that compared to LCGL, Δ ​ There was no significant difference in spore morphology. ​ A), Explanation ​Gene deletion does not affect spore formation. Measurement of LCGL and Δ... ​ The bacterial cell dry weight was measured, and a corresponding change curve was plotted. The results showed Δ ​ The biomass difference compared to LCGL is not significant. ​ B), implying ​ The deletion of the gene did not affect the primary metabolism of the bacteria.

[0058] 3. Δ ​ Transcriptional analysis of related genes

[0059] qRT-PCR data showed that, compared with LCGL, Δ ​ The transcriptional levels of regulatory genes, resistance genes, and most structural genes within the lincomycin biosynthesis gene cluster were upregulated to varying degrees. ​ This indicates that SLCG_3615 negatively regulates the transcriptional level of genes within the cluster, thereby controlling the biosynthesis of lincomycin.

[0060] 4. Knockout in high-yielding strain LA219X ​ Genes can increase lincomycin production.

[0061] LA219XΔ 3615 The strain and the high-yielding strain LA219X were plated and activated, then inoculated into shake flasks of industrial seed culture medium and cultured at 30℃ and 220rpm for 48h. Afterward, they were transferred to fermentation medium and cultured for another 168h. After fermentation, extraction and concentration were performed, and HPLC analysis showed that compared to LA219X, LA219XΔ 3615 The yield of lincomycin can be increased by 13.4% ( ​ This indicates that in the high-yielding strain LA219X ​ Genes are also involved in controlling lincomycin production.

[0062] 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_3615 A method for increasing lincomycin production through gene therapy, characterized in that, Knockout of TetR family transcriptional regulatory genes in Streptomyces lincosae via genetic engineering SLCG_3615 A high-yield engineered strain of lincomycin was obtained, and the obtained strain was used to produce lincomycin through fermentation; among which, SLCG_3615 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The method according to claim 1, characterized in that, The target gene-engineering method for knockout is Streptomyces lincosinate LA219X. SLCG_3615 Gene.

3. The method according to claim 1, characterized in that, The SLCG_3615 The amino acid sequence encoded by the gene is shown in SEQ ID NO.

2.

4. The method according to claim 1, characterized in that, The SLCG_3615 Gene products negatively regulate lincomycin biosynthesis.

5. A kind SLCG_3615 The application of gene knockout modified Streptomyces lincosinate is characterized by, The SLCG_3615 The gene knockout modified Streptomyces lincomycin was constructed by the method described in any one of claims 1 to 4 and is used for the fermentation production of lincomycin.

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