A method for improving the production of lincomycin by modifying streptomyces lincolnensis slcg_1540 gene and application

Abstract

This invention provides a method for modifying Streptomyces lincosinate. SLCG_1540 Methods to increase lincomycin production through gene therapy involve knocking out LacI family transcriptional regulatory genes in Streptomyces lincomyces via genetic engineering. SLCG_1540 A high-yield engineered strain of lincomycin was obtained, and the obtained strain was used to produce lincomycin through fermentation; among which, SLCG_1540 The nucleotide sequence of the gene is shown in SEQ ID NO.1. This invention also provides the above-mentioned... SLCG_1540 Applications of gene knockout modified *Streptomyces lincomycin*. The advantages of this invention are: the screening of a negative regulator of lincomycin biosynthesis, SLCG_1540, and experimental findings that gene knockout of *Streptomyces lincomycin* chromosomes via genetic engineering... SLCG_1540 This invention provides technical support for increasing lincomycin fermentation yield in industrial production by generating genes that can produce high-yield strains of lincomycin.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering, and particularly relates to a method for improving lincomycin yield by modifying Streptomyces lincolnensis SLCG_1540 and application thereof. BACKGROUND

[0002] As an important source of secondary metabolites in the microbial world, Streptomyces can synthesize a variety of bioactive compounds, covering antifungal, antiviral, antitumor, antihypertensive and other functional types. Among them, antibiotics and immunosuppressants are most widely used in clinical medicine and industrial production. With the rapid progress of whole genome sequencing technology, more and more genome sequences of Streptomyces have been successfully determined and in-depth analyzed. Research has found that although the genome contains a large number of secondary metabolite biosynthetic gene clusters, most of the gene clusters are in a silent state for a long time, or can only express at a low level to synthesize a small amount of secondary products, which greatly limits the development of the potential application value of Streptomyces.

[0003] In the industrial production of Streptomyces related products, the acquisition of high-yield strains is the core link to improve product yield and reduce production cost. Traditionally, high-yield strains are mainly obtained by physical or chemical mutagenesis. This method causes gene mutations by randomly damaging the genome of the strain, and then obtains possible high-yield mutant strains through a large number of screening. However, this process has obvious limitations: on the one hand, the mutagenesis effect is random, and it is impossible to direct modification of the target metabolic pathway, resulting in extremely low screening efficiency, often requiring a large amount of time and manpower; on the other hand, the mutagenesis mechanism lacks clear theoretical support, making it difficult to explain the molecular regulation principle of high-yield traits, and unable to provide systematic guidance for subsequent strain optimization.

[0004] Lincomycin, as a lincomycin amide antibiotic specifically synthesized by Streptomyces lincolnensis, together with its derivative clindamycin, constitutes an important drug for the clinical treatment of Gram-positive and some Gram-negative bacterial infections. The mechanism of action of the two is clear. They specifically bind to the peptidyl transferase center region of 23s rRNA in the 50s subunit of bacterial ribosomes, hinder the elongation process of the peptide chain, and then inhibit the synthesis of proteins in bacteria, ultimately achieving the effect of inhibiting bacterial growth and playing an antibacterial role. In clinical applications, lincomycin is widely used to treat bone and joint infections due to its high efficacy, low toxicity and difficulty in producing cross-resistance with other antibiotics. It also plays an irreplaceable role in preventing postoperative intramembranous infections, oral infections, skin and mucous membrane infections, etc., and has a high clinical value and broad market application prospect. Therefore, it is of great significance to carry out directional breeding research on Streptomyces lincolnensis to improve the yield of lincomycin and promote the development of related pharmaceutical industries.

[0005] LacI family transcriptional regulators (LacI-TFs) are a class of DNA-binding proteins widely present in bacteria, with the LacI repressor protein in Escherichia coli as a typical representative, and its homologous proteins are classified into the LacI / GalR family. At present, thousands of family members have been identified in different groups of bacteria. The transcriptional regulators play a key role in gene expression regulation, and are usually structurally composed of a DNA-binding domain and a regulatory binding domain, which are connected by a linker. Functionally, when the effector molecule binds to the regulatory binding domain, it will trigger a conformational change, thereby changing the binding affinity of the DNA, and achieving precise regulation of the expression of target genes. Essentially, the LacI family transcriptional regulator is equivalent to a "molecular switch" in the bacterial body, which can closely link the metabolic state of the bacterial body with the gene expression level, helping the bacteria to adapt to the changes in nutrients in the environment, and ensuring the normal growth and metabolic activities of the bacteria.

[0006] Studies have shown that the regulation range of LacI family transcriptional regulators is wide, and they not only participate in the expression regulation of basic metabolism related genes such as sugar utilization pathways, but also regulate various biological processes such as amino acid biosynthesis, antibiotic resistance, and independent factor production of bacteria. For Streptomyces lincolnensis, the sugar metabolism pathway is the core link of its growth and development and secondary metabolism: on the one hand, sugar metabolism provides essential energy and material basis for the growth of the bacterial body; on the other hand, the intermediates of sugar metabolism can directly participate in the biosynthesis process of lincomycin, and by regulating the sugar metabolism flux, the synthesis efficiency of lincomycin can be directly affected, thereby having a key influence on the fermentation yield and product quality.

[0007] Therefore, in-depth study on the function of LacI family transcriptional regulators in Streptomyces lincolnensis is expected to provide new molecular targets and theoretical basis for the directional modification of lincomycin synthesis pathway and the construction of high-yield engineering strains. SUMMARY

[0008] The technical problem to be solved by the present application is to provide a method for improving the yield of lincomycin by modifying the gene of Streptomyces lincolnensis and application. SLCG_1540

[0009] The present application solves the above technical problems by adopting the following technical solutions:

[0010] A method for improving the yield of lincomycin by modifying the gene of Streptomyces lincolnensis, which comprises the following steps: SLCG_1540 knocking out the LacI family transcriptional regulator gene of Streptomyces lincolnensis by genetic engineering, SLCG_1540 obtaining a high-yield engineering strain of lincomycin, and fermenting lincomycin with the obtained strain; wherein, SLCG_1540 the nucleotide sequence of the gene is shown in SEQ ID NO. 1. ​

[0011] As one of the preferred embodiments of the present invention, the target of the gene engineering approach for knockout is Streptomyces lincosum LA219X. SLCG_1540 Gene.

[0012] As one of the preferred embodiments of the present invention, the SLCG_1540 The amino acid sequence encoded by the gene is shown in SEQ ID NO.2.

[0013] As one of the preferred embodiments of the present invention, the SLCG_1540 Gene products negatively regulate lincomycin biosynthesis.

[0014] A sort of SLCG_1540 The application of gene knockout modified Streptomyces lincosinate, the aforementioned SLCG_1540 Gene knockout modified Streptomyces lincomyces was constructed using the above method and used for the fermentation production of lincomycin.

[0015] The advantages of this invention compared to the prior art are:

[0016] In this study, the negative regulator of lincomycin biosynthesis, SLCG_1540, was screened and deleted from the chromosome of Streptomyces lincomycin through genetic engineering. SLCG_1540 The gene can be used to obtain high-yield strains of lincomycin, providing technical support for increasing the fermentation yield of lincomycin in industrial production.

[0017] Knockout in Streptomyces lincosae LCGL SLCG_1540 The gene, SLCG_1540, increased lincomycin production by 16.8%, indicating that SLCG_1540 is a negative regulator of lincomycin biosynthesis. Using the high-yielding strain LA219X as the starting strain, the gene was knocked out on its chromosome. SLCG_1540 The gene knockout increased lincomycin production by 11%, indicating that the gene knockout... SLCG_1540 The techniques for increasing lincomycin yield are also applicable to high-yield industrial strains. Attached Figure Description

[0018] Figure 1 yes SLCG_1540 A diagram showing the location of a gene and its neighboring genes on a chromosome;

[0019] Figure 2 It is Δ SLCGL_1540 Construct a schematic diagram;

[0020] Figure 3 It is Δ SLCGL_1540 PCR identification results (in the figure, M: 5000 bp DNA Marker; +: pKC1139-Δ1540; -: LCGL; 1-2: Δ SLCGL_1540 );

[0021] Figure 4 It is Δ SLCGL_1540 PCR identification results of the / pIB139-1540 reversion strain and the LCGL / pIB139-1540 overexpression strain (in the figure, the PCR product is the Apr resistance gene, 801bp; M: 5000 bp DNA Marker; +: pIB139 plasmid; -: ultrapure water; lane 1: Δ SLCGL_1540 / pIB139-1540 re-expression strain; lanes 2-3: LCGL / pIB139-1540 overexpression strain).

[0022] Figure 5 It is Δ SLA219X_1540 PCR identification results of the strain (in the figure, M: 5000 bp DNA Marker; +: pKC1139-Δ1540 plasmid; -: ultrapure water; lanes 1-3: Δ SLA219X_1540 (deleted strain);

[0023] Figure 6 The originating strains are LCGL and Δ SLCGL_1540 Analysis of lincomycin production (in the figure, "**") P < 0.01);

[0024] Figure 7 yes SLCG_1540 The effect of genes on strain morphological differentiation and Δ SLCGL_1540 Biomass measurement (in the figure, Figure A shows LCGL and Δ) SLCGL_1540 Spore growth of the strain, where, left: Δ SLCGL_1540 Right: LCGL strain; Figure B shows LCGL and Δ SLCGL_1540 Results of mycelial dry weight determination of the strain).

[0025] Figure 8 It is LCGL, Δ SLCGL_1540 Δ SLCGL_1540 Lincomycin production analysis of strains / pIB139-1540 and LCGL / pIB139-1540 (in the figure, "*") P <0.1, "**": P < 0.01);

[0026] Figure 9 This is an analysis of the transcriptional level of genes related to the lincomycin biosynthesis gene cluster (as shown in the figure: *). P <0.1, "**": P < 0.01, "***": P <0.001);

[0027] Figure 10 It is a high-yield lincomycin strain LA219X, Δ SLA219X_1540Analysis of lincomycin production (in the figure, "***") P <0.001). Detailed Implementation

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] Table 1. This invention relates to strains and plasmids.

[0033]

[0034] Table 2 This invention relates to primers.

[0035]

[0036] Example 1 SLCG_1540 Gene-related information:

[0037] SLCG_1540 For the location of the gene and its neighboring genes on the chromosome, see [link to relevant documentation]. Figure 1 .

[0038] According to LCGL genome information, SLCG_1540 The gene is 1041 bp long and the protein monomer size is 36.8 kDa.

[0039] Specifically, SLCG_1540 The specific 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.

[0040] Example 2 SLCG_1540 Construction of gene deletion mutants ( Figure 2 ):

[0041] Using the LCGL genome as a template, 1540-P1 、 1540-P2 、 1540-P3 、 Primers 1540-P4 were used to amplify the following results: SLCG_1540 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 / Eco RI Xba I / Hind III. The upstream and downstream homologous arms were digested and recovered using enzymes, and then quantified. 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 in a 1:7:7 ratio and ligated overnight at 16°C with T4 ligase. 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-Δ1540 plasmid was then extracted.

[0042] The obtained pKC1139-Δ1540 plasmid was transformed into LCGL protoplasts via PEG3350-mediated transformation, and homologous large fragment recombination technology was used to complete the transformation. SLCG_1540Construction of the knockout strain. The specific experimental procedure was as follows: 4000 ng pKC1139-Δ1540 was mixed with 50 μL of LCGL protoplasts, then 200 μL of PEG3350 was added. After standing for 5 min, the mixture was diluted and spread onto R5 plates, and incubated at 30℃ for approximately 20 h. When membranous cells grew on the surface, Apr antibiotic was added for screening. After approximately 4 days, single colonies with blackened bottoms grew on the plates. These were enriched on industrial plates containing Apr and incubated at 30℃ for 3 days. Subsequently, the enriched spores were picked, diluted, and spread onto antibiotic-free R5 plates, and incubated at 37℃ for 3 days to induce plasmid loss. Finally, a portion of the single colonies from the R5 plates were plated onto Apr industrial plates, and the remaining single colonies were plated onto antibiotic-free industrial plates. Single 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 15 minutes. PCR identification was then performed using primers 1540-P5 and 1540-P6. The results are as follows: Figure 3 As shown, the correct knockout strain Δ was obtained. SLCGL_1540 .

[0043] Example 3 SLCG_1540 Gene reversion and construction of overexpression strains:

[0044] Using the LCGL genome as a template, primers 1540-P7 and 1540-P8 were used to amplify genomes with chromosomes at both ends. Nde I and Xba I restriction site SLCG_1540 Gene fragments are extracted and recycled. Used. Nde I and Xba I double enzyme digestion SLCG_1540 The fragment and pIB139 plasmid were recovered and, under 16°C, T4 ligase was added to ligate them. SLCG_1540 The pIB139 plasmid was ligated and transformed into *E. coli* DH5α. The cells were plated on LB agar plates containing Apr antibodies and incubated at 37°C for approximately 12 hours, after which single colonies grew. Single colonies were selected and transferred to liquid LB medium for 6 hours of culture, followed by colony PCR identification. Correctly identified colonies were sequenced, and after successful sequencing, the pIB139-1540 plasmid was obtained through preservation.

[0045] pIB139-1540 plasmid was introduced into LCGL and Δ through protoplasmic transformation, respectively. SLCG_1540 In protoplasts. 20 hours after transformation, the culture medium was covered with apramycin aqueous solution and cultured until transformants grew. Transformants were picked and spread onto industrial plates containing Apr resistance. After Apr-resistant transformants grew, they were verified using primers Apr-F / R. Figure 4 As shown, the correct Δ is obtained. SLCGL_1540 / pIB139-1540 (replacing strain), LCGL / pIB139-1540 strain (overexpressing strain).

[0046] Example 4: Construction of LA219X / pKC1139-Δ1540:

[0047] The pKC1139-Δ1540 plasmid was transformed into LA219X protoplasts via PEG3350 to construct the LA219X / pKC1139-Δ1540 strain. The screening method was as described in Example 2, and the PCR identification results are as follows. Figure 5 .

[0048] Example 5: HPLC detection of Streptomyces lincosum fermentation products:

[0049] 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.

[0050] Example 6: Detection of *Streptomyces lincosum* mycelial biomass:

[0051] LCGL and Δ were inoculated at the same amount. SLCGL_1540 Inoculate into 5 mL of liquid TSBY, incubate at 30°C on a shaker for 48 h, then take equal amounts of LCGL and SLCGL_1540 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 SLCGL_1540 The cell dry weight of LCGL was used to plot the cell biomass curve based on the growth time.

[0052] Example 7, Δ SLCG_1540 Transcriptional analysis of related genes in the middle:

[0053] Collect LCGL and Δ over 24 hours SLCG_1540 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.

[0054] Example 8: Analysis of the results of the above examples:

[0055] 1. SLCG_1540 negatively regulates the biosynthesis of lincomycin.

[0056] Δ SLCGL_1540 After fermentation in the fermentation medium for 168 hours, the lincomycin yield was detected by HPLC, and Δ was found to be... SLCGL_1540 Production increased by approximately 16.8% compared to LCGL. Figure 6 This has been confirmed. SLCG_1540 It is a negative regulatory gene for lincomycin biosynthesis in *Streptomyces lincosae*, and its inactivation in *Streptomyces lincosae* is achieved through genetic engineering. SLCG_1540 Genes can increase lincomycin production.

[0057] 2. Missing SLCG_1540 The Influence of Genes on Spore Morphology Differentiation and Cell Growth

[0058] In order to determine SLCG_1540 Whether the gene regulates the spore formation of the bacterial cell, and whether LCGL and Δ SLCGL_1540 Simultaneously, the strain was plated on MGM plates and incubated at 30°C for 7 days, with daily observation of spore growth. Results showed that compared to LCGL, Δ SLCGL_ 1540 There was no significant difference in spore morphology. Figure 7 A), Explanation SLCG_1540 Gene deletion does not affect spore formation. Measurement of LCGL and Δ... SLCGL_1540 The bacterial cell dry weight was measured, and a corresponding change curve was plotted. The results showed Δ SLCGL_1540 The biomass difference compared to LCGL is not significant. Figure 7 B), implying SLCG_1540 The deletion of the gene did not affect the primary metabolism of the bacteria.

[0059] 3. SLCG_1540 Gene reversion and overexpression

[0060] To further verify Δ SLCGL_1540 The increase in lincomycin production is due to SLCG_1540 Caused by gene deletion, SLCG_1540 Gene expression vector pIB139-1540 was introduced into LCGL and Δ SLCGL_1540 The revertant strain Δ was obtained from the protoplasts via PCR identification. SLCGL_1540 / pIB139-1540 and overexpression strain LCGL / pIB139-1540. LCGL and Δ SLCGL_1540 A series of strains were subjected to shake-flask fermentation, and HPLC analysis showed that: Δ SLCGL_1540The yield of lincomycin increased by 16.8% compared to LCGL; the yield of the reversion strain Δ SLCGL_1540 The yield of lincomycin in / pIB139-1540 recovered to the level of lincomycin in LCGL; the yield of lincomycin in LCGL / pIB139-1540 was 13.5% lower than that in LCGL. Figure 8 These results indicate that SLCG_1540 negatively regulates the biosynthesis of lincomycin.

[0061] 4. Δ SLCG_1540 Transcriptional analysis of related genes

[0062] qRT-PCR data showed that, compared with LCGL, Δ SLCGL_1540 The transcriptional levels of regulatory genes, resistance genes, and most structural genes within the lincomycin biosynthesis gene cluster were upregulated to varying degrees. Figure 9 This indicates that SLCG_1540 negatively regulates the transcriptional level of genes within the cluster, thereby controlling the biosynthesis of lincomycin.

[0063] 5. Knockout in high-yielding strain LA219X SLCG_1540 Genes can increase lincomycin production.

[0064] Δ SLA219X_1540 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, Δ... SLA219X_1540 Lincomycin production increased by 11% ( Figure 10 This indicates that the high-yielding strain LA219X... SLCG_1540 Genes are also involved in controlling lincomycin production.

[0065] 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 increasing lincomycin production by Streptomyces lincolnensis by genetic engineering. SLCG_1540 Knocking out lacI family transcription regulation gene in streptomyces lincolnensis through genetic engineering approach SLCG_1540 , obtaining lincomycin high-yield engineering strain, and fermenting lincomycin using the obtained strain; wherein, SLCG_1540 The nucleotide sequence of the gene is shown in SEQ ID NO.

1. ​ 2. The method of claim 1, wherein, The object of the genetic engineering approach is to knock out the SLCG_1540 gene in Streptomyces lincolnensis LA219X.

3. The method of claim 1, wherein, The SLCG_1540 The amino acid sequence encoded by the gene is shown in SEQ ID NO.

2.

4. The method of claim 1, wherein, The SLCG_1540 The gene product negatively regulates lincomycin biosynthesis.

5. A kind SLCG_1540 The application of gene knockout modified Streptomyces lincosinate is characterized by, The SLCG_1540 The genetically modified Streptomyces lincolnensis is obtained by the method of any one of claims 1-4, and is used for fermentation production of lincomycin.

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