Method for improving lincomycin yield by transforming streptomyces lincomycin SLCG3904 gene and application

Abstract

The invention provides a method for improving the yield of lincomycin by modifying a streptomyces lincomycin SLCG3904 gene, which comprises the following steps: deleting a GlnR family transcriptional regulation gene SLCG3904 in the streptomyces lincomycin through a genetic engineering way to obtain a lincomycin high-yield engineering strain, and fermenting the obtained strain to produce the lincomycin. Wherein the nucleotide sequence of the SLCG3904 gene is as shown in SEQ ID NO. 1. The invention further provides application of the method. The invention has the advantages that the lincomycin biosynthesis negative regulator SLCG3904 (GlnR family) is screened in the research, the SLCG3904 gene on the streptomyces lincomycin chromosome is deleted through a genetic engineering way, a lincomycin high-yield strain can be obtained, and a technical support is provided for improving the lincomycin fermentation yield 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 for increasing the yield of lincomycin by transforming the gene of Streptomyces lincomycin SLCG_3904. Background Art

[0002] Streptomyces is an important source of secondary metabolites and can produce secondary metabolites with a variety of biological activities, such as antifungal, antiviral, antitumor, and antihypertensive compounds, among which the most important are antibiotics and immunosuppressants.

[0003] Lincomycin, a lincosamide antibiotic produced mainly by Streptomyces lincomycin, and its derivative clindamycin are both very important antibiotics in clinical practice. They act on Gram-positive bacteria and some Gram-negative bacteria. Their action site is the peptidyl transferase center of bacterial ribosomes. They inhibit the elongation of peptide chains by binding to the central ring of 23srRNA in the 50s subunit, inhibit protein synthesis in bacteria, and thus affect bacterial growth. Lincomycin is commonly used to treat bone and joint infections, and can also prevent postoperative intramembranous infections, oral infections, skin and mucosal infections, etc. It has high efficacy, but low toxicity, and is not easy to produce cross-resistance. It is a class of antibiotics with broad application prospects.

[0004] With the rapid progress of whole genome sequencing technology, more and more lincomycin genome sequences have been determined and analyzed, but it is found that most of its biosynthetic gene clusters are in a silent state or the secondary product synthesis ability is weak. 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 lincomycin or intermediate product production.

[0005] GlnR (Glutamine Synthetase Regulatory Protein) is widely present in bacteria and archaea. It is a global transcriptional regulatory protein in bacteria. It plays a key role in many bacteria and is mainly involved in the regulation of nitrogen metabolism. Studies have found that GlnR can adapt to environmental changes by directly regulating multiple genes related to nitrogen metabolism, such as urea metabolism and nitrate reduction. GlnR, a global regulator of nitrogen metabolism, plays a key role in the process of nitrate reduction. GlnR can promote the transcription of nitrate-specific transporter genes, thereby increasing the concentration of nitrate in bacteria. In addition, GlnR also increases the accumulation of glutamate in cells by promoting the transcription level of related genes in the nitrate assimilation pathway. Glutamate can form tyrosine, a precursor substance for the synthesis of lincomycin, through transamination. At the same time, studies have found that when Streptomyces lincomycin is carrying out antibiotic biosynthesis, the addition of a certain amount of nitrate will affect the primary metabolism of antibiotics and further affect the process of antibiotic production.

[0006] Based on this, the present invention screened the GlnR family SLCG_3904 gene in Streptomyces lincomycin, and attempted to transform the SLCG_3904 gene of Streptomyces lincomycin to increase the yield of lincomycin. Summary of the invention

[0007] 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_3904.

[0008] The present invention adopts the following technical solutions to solve the above technical problems:

[0009] A method for improving the yield of lincomycin by modifying the SLCG_3904 gene of Streptomyces lincomycin, wherein the GlnR family transcriptional regulatory gene SLCG_3904 in Streptomyces lincomycin is deleted through genetic engineering to obtain a lincomycin high-yield engineered strain, and the obtained strain is used to ferment and produce lincomycin; wherein the nucleotide sequence of the SLCG_3904 gene is shown in SEQ ID NO.1.

[0010] As one of the preferred embodiments of the present invention, the amino acid sequence encoded by the SLCG_3904 gene is shown in SEQ ID NO.2.

[0011] As one of the preferred embodiments of the present invention, the SLCG_3904 gene product negatively regulates lincomycin biosynthesis.

[0012] An application of the method is to knock out the SLCG_3904 gene in an industrial strain to obtain a high-yield mutant for lincomycin production.

[0013] As one of the preferred embodiments of the present invention, the industrial strain is specifically selected as the high-yield strain LA219X.

[0014] The advantages of the present invention compared to the prior art are:

[0015] In the present invention, the negative regulator of lincomycin biosynthesis SLCG_3904 (GlnR family) was screened. By deleting the SLCG_3904 gene on the chromosome of Streptomyces lincomycin through genetic engineering, a high-yield strain of lincomycin can be obtained, providing technical support for increasing the fermentation yield of lincomycin in industrial production.

[0016] Specifically, knocking out the SLCG_3904 gene in Streptomyces lincomycin LCGL increased the lincomycin yield by 18.2%, indicating that SLCG_3904 is a negative regulator involved in the biosynthesis of lincomycin. Using the high-yield strain LA219X as the starting strain, knocking out the SLCG_3904 gene on its chromosome increased the lincomycin yield by 12.8%, indicating that the technology of knocking out SLCG_3904 to increase lincomycin yield is also applicable to industrial high-yield strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the location information map of the SLCG_3904 gene and its neighboring genes on the chromosome;

[0018] Figure 2 is a schematic diagram of the construction of ΔSLCGL_3904;

[0019] Figure 3 is the PCR identification result of ΔSLCGL_3904 (in the figure, “M”: 5000 bp DNA Marker; “+”: pKC1139-Δ3904; “-”: LCGL; “1”: ΔSLCGL_3904);

[0020] Figure 4 It is the PCR identification result of ΔSLCGL_3904 / pIB139-3904 complementation and LCGL / pIB139-3904 overexpression (in the figure, "M": 5000bp DNA Marker; "+": pIB139 plasmid; "-": ultrapure water; "1": ΔSLCGL_3904 / pIB139-3904 complementation strain; "2": LCGL / pIB139-3904 overexpression strain; PCR product is Apr resistance gene, 776bp);

[0021] Figure 5This is the PCR identification result of the lincomycin high-producing strain ΔSLA219X_3904 (in the figure, “M”: 5000 bp DNA Marker; “+”: pKC1139-Δ3904 plasmid; “-”: LCGL; “1”: ΔSLA219X_3904);

[0022] Figure 6 is the analysis of lincomycin production of the starting strain LCGL and ΔSLCGL_3904 (in the figure, “*”: P < 0.1);

[0023] Figure 7 is the effect of SLCG_3904 gene on strain morphological differentiation and the determination of ΔSLCGL_3904 biomass (in the figure, Figure A shows the spore growth of LCGL and ΔSLCGL_3904 strains, where left: LCGL strain, right: ΔSLCGL_3904; Figure B shows the determination of mycelium dry weight of LCGL and ΔSLCGL_3904 strains);

[0024] Figure 8 This is the analysis of lincomycin production of LCGL, ΔSLCGL_3904, ΔSLCGL_3904 / pIB139-3904, and LCGL / pIB139-3904 strains (in the figure, "*": P<0.1, "**": P<0.01, "ns": no significant difference).

[0025] Fig. 9 is the analysis of transcriptional levels of genes related to the lincomycin biosynthesis gene cluster (in the figure, “*”: P < 0.1, “**”: P < 0.01, “ns”: no significant difference);

[0026] Fig.10 This is the analysis of lincomycin production in the lincomycin high-producing strains LA219X and ΔSLA219X_3904 (in the figure, "***": P<0.001). DETAILED DESCRIPTION

[0027] 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.

[0028] 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 a strain obtained by improving 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 240bp base sequence containing 4 attBΦC31 site sequences, and the base sequence is shown in SEQ ID NO.3.

[0029] 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 was cultured in tryptone soy broth (TSBY) medium at 30°C or on modified Gould's 1 (MGM) plates containing 1.8% agar.

[0030] 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 a reagent company. 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.

[0031] Table 1 The present invention relates to strains and plasmids

[0032]

[0033]

[0034] Table 2 The present invention relates to primers

[0035]

[0036]

[0037]

[0038] Example 1

[0039] SLCG_3904 gene related information:

[0040] The location of SLCG_3904 gene and its neighboring genes on chromosomes can be found in Figure 1 .

[0041] The specific nucleotide sequence of the SLCG_3904 gene is shown in SEQ ID NO.1, with a length of 780 bp, the encoded amino acid sequence is shown in SEQ ID NO.2, and the protein monomer size is about 28 KDa.

[0042] Example 2

[0043] Construction of SLCG_3904 gene deletion mutant (see Figure 2 ):

[0044] Using the LCGL genome as a template and 3904-P1, 3904-P2, 3904-P3, and 3904-P4 as primers, 1.5 kb of the upstream and downstream homology arms of SLCG_3904 were amplified and recovered. At 37°C, the upstream and downstream homology arms were digested and recovered and quantified using XbaI / EcoRI and XbaI / HindIII, respectively. While digesting the homology arms, the pKC1139 plasmid was double digested with EcoRI / HindIII and then recovered and quantified. According to the quantitative results, the digested plasmid, upstream homology arms, and downstream homology arms were mixed at a ratio of 1:10:10, and T4 ligase was added at 22°C for ligation. Then, all the ligation products were transformed into Escherichia coli DH5α, diluted and coated on an LB plate with apramycin (Apr) resistance and cultured for about 12 hours at 37°C until a single clone grew. Finally, a single clone was picked and cultured in LB liquid medium containing Apr resistance, and then transferred to 5 mL of LB medium containing Apr resistance and cultured for 12 hours to extract the pKC1139-Δ3904 plasmid.

[0045] The obtained pKC1139-Δ3904 plasmid was transformed into the protoplasts of LCGL by PEG3350, and the SLCG_3904 knockout strain was constructed by homologous large fragment recombination technology. The specific operations are as follows:

[0046] Take 4000ng of pKC1139-Δ3904 and mix it with 50μL of LCGL protoplasts, then add 200mL PEG3350 and let it stand for 5min. Dilute the mixture and spread it on the R5 plate, and culture it at 30℃ for 20h; wait for the film-like bacteria to grow on its surface, and add Apr antibiotics for covering screening. About 4d later, a single clone with a black bottom grows on the plate, which is enriched on the industrial plate containing Apr and cultured at 30℃ for about 3d. Subsequently, pick the enriched spores, dilute and spread them on the R5 plate without resistance, and culture them at 37℃ for 3d to lose the plasmid. Finally, take some monoclonal colonies on the R5 plate and spread them on the Apr industrial plate, and the remaining single colonies on the non-antibiotic industrial plate. Select the monoclonal clones that cannot grow on the Apr plate but can grow on the non-antibiotic plate and culture them until spores are produced, then scrape a certain amount of spores into sterile water and boil them at high temperature for 15min. Then PCR identification was performed using 3904-P5 and 3904-P6 as primers. Figure 3 As shown, the correct knockout strain ΔSLCGL_3904 was obtained.

[0047] Example 3

[0048] Construction of SLCG_3904 gene complementation and overexpression strains:

[0049] Using the LCGL genome as a template, 3904-P7 and 3904-P8 as primers, the SLCG_3904 gene fragment with NdeI and XbaI restriction sites at both ends was amplified and recovered. The SLCG_3904 fragment and the pIB139 plasmid were double-digested with NdeI and XbaI and recovered. At 22°C, T4 ligase was added to connect the SLCG_3904 and pIB139 plasmids, and transformed into Escherichia coli DH5α, and spread on an LB plate with Apr resistance at 37°C for about 12 hours to grow a single clone. After the single clone was transferred to liquid LB culture medium and cultured for 5 hours, PCR identification of the bacterial liquid was performed. The correctly identified bacteria were sent for sequencing, and after sequencing was correct, the pIB139-3904 plasmid was obtained by seed preservation.

[0050] The pIB139-3904 plasmid was introduced into LCGL and ΔSLCG_3904 protoplasts by protoplast transformation. 20 h after transformation, the culture medium was covered with an aqueous solution of apramycin and cultured until transformants grew out. Transformants were selected and spread on industrial plates with Apr resistance. After the Apr-resistant transformants grew out, the primers Apr-F / R were used for verification. Figure 4 As shown, the correct ΔSLCGL_3904 / pIB139-3904 and LCGL / pIB139-3904 strains were obtained.

[0051] Example 4

[0052] Construction of lincomycin high-producing strain ΔSLA219X_3904:

[0053] The pKC1139-Δ3904 plasmid was transformed into the protoplasts of LA219X via PEG3350 to construct the ΔSLA219X_3904 strain. The construction and screening method were referenced to Example 3. The PCR identification results were as follows: Figure 5 .

[0054] Example 5

[0055] HPLC detection of fermentation products of Streptomyces lincomyces:

[0056] After culturing Streptomyces lincosus on the slant medium for 7 days, 1 cm 2 The spore mass was inoculated into the seed culture medium. After shaking culture at 30℃ and 240rpm for 48h, it was transferred to the fermentation medium and shaken at 30℃ and 240rpm for 7d. Then, 2mL of bacterial solution was centrifuged at 12000rpm for 10min, and 200μL of supernatant was mixed with 800μL of anhydrous ethanol and centrifuged at 12000rpm for 10min. Finally, the supernatant was injected into the test bottle through an organic filter membrane for yield detection.

[0057] Example 6

[0058] Streptomyces lincomyces mycelium biomass detection:

[0059] LCGL and ΔSLCGL_3904 were inoculated into 5 mL of liquid TSBY with the same inoculation amount, and cultured at 30°C in a shaking incubator for 48 h. Take an equal amount of fresh bacterial solution of LCGL and ΔSLCGL_3904 in YMG medium and culture at 30°C and 240 rpm for 7 days; during this period, 1 mL of fresh bacterial solution was taken out every 24 hours and stored in a -20°C refrigerator. After the fermentation was completed, the 7-day sample was centrifuged at 12000 rpm for 10 min and the supernatant was discarded. Add 1 mL of anhydrous ethanol to wash the cells, centrifuge at 12000 rpm for 10 min, and discard the supernatant. Place the moist cells in a 65°C oven for 2 days, weigh them, and record the cell mass. The cell dry weight of ΔSLCGL_3904 and LCGL was plotted against the growth time to draw the cell biomass curve.

[0060] Example 7

[0061] Transcription analysis of related genes in ΔSLCG_3904:

[0062] The LCGL and ΔSLCG_3904 bacterial cultures were collected for 24 hours, and the required RNA was obtained using the Total Gold RNA Extraction Kit. After being reversed into cDNA, it was detected using a real-time fluorescence quantitative PCR instrument.

[0063] Example 8

[0064] Analysis of the results of the above embodiments:

[0065] 1. SLCG_3904 positively regulates the biosynthesis of lincomycin

[0066] The SLCG_3904 gene deletion mutant was confirmed by PCR identification ( Figure 3 After ΔSLCGL_3904 was fermented in the fermentation medium for 168 h, the lincomycin production was detected by HPLC, and it was found that the production of ΔSLCGL_3904 increased by about 18.2% compared with that of LCGL ( Figure 6 ). The above results confirmed that SLCG_3904 is a negative regulatory gene for lincomycin biosynthesis in Streptomyces lincomycin, and inactivating the SLCG_3904 gene in Streptomyces lincomycin through genetic engineering can increase the lincomycin production.

[0067] 2. Effect of SLCG_3904 gene deletion on spore morphological differentiation and bacterial growth

[0068] In order to determine whether the SLCG_3904 gene regulates the spore formation of bacteria, LCGL and ΔSLCGL_3904 were simultaneously plated on MGM plates and cultured at 30°C for 7 days, and the spore growth of the strains was observed daily. The results showed that compared with LCGL, the spore morphology of ΔSLCGL_3904 was not significantly different ( Figure 7 A), indicating that the deletion of the SLCG_3904 gene does not affect spore formation. At the same time, the dry weight of LCGL and ΔSLCGL_3904 was measured, and the corresponding change curve was drawn. The results showed that the biomass of ΔSLCGL_3904 was not much different from that of LCGL ( Figure 7 B), suggesting that the loss of SLCG_3904 gene did not affect the primary metabolism of the bacteria.

[0069] 3. SLCG_3904 gene complementation and overexpression

[0070] In order to further verify that the increase in lincomycin production in ΔSLCGL_3904 was caused by the deletion of the SLCG_3904 gene, the SLCG_3904 gene expression vector pIB139-3904 was introduced into the protoplasts of LCGL and ΔSL C GL_3904, respectively. After PCR identification, the complemented strain ΔSLCGL_3904 / pIB139-3904 and the overexpression strain LCGL / pIB139-3904 were obtained. The LCGL and ΔSLCGL_3904 series strains were fermented in shake flasks. The HPLC test results showed that the lincomycin production in ΔSLCGL_3904 was 18.2% higher than that in LCGL; the lincomycin production in the complemented strain ΔSLCGL_3904 / pIB139-3904 was restored to the level of lincomycin in LCGL; the lincomycin production in LCGL / pIB139-3904 was 16.6% lower than that in LCGL ( Figure 8 These results suggest that SLCG_3904 negatively regulates lincomycin synthesis.

[0071] 4. Transcription analysis of related genes in ΔSLCG_3904

[0072] 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_3904 were upregulated to varying degrees ( Fig. 9 ), indicating that SLCG_3904 negatively regulates the transcription level of genes in the cluster and thus controls the biosynthesis of lincomycin.

[0073] 5. Knocking out the SLCG_3904 gene in the high-yield strain LA219X can increase the yield of lincomycin

[0074] The ΔSLA219X_3904 strain and the high-yield strain LA219X were plated and activated, and then inoculated into shake flasks of industrial seed culture medium and cultured at 30°C and 220rpm for 48h. They were transferred to fermentation medium and cultured for 168h. After fermentation, the extracts were concentrated and analyzed by HPLC. Compared with LA219X, the lincomycin yield of ΔSLA219X_3904 increased by 12.8% ( Fig.10 ), which indicates that the SLCG_3904 gene in the high-yield strain LA219X is also involved in controlling the production of lincomycin.

[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 in the protection scope of the present invention.

Claims

1. A method for increasing the yield of lincomycin by modifying the gene of Streptomyces lincomycin SLCG_3904, characterized in that: The GlnR family transcriptional regulatory gene SLCG_3904 in Streptomyces lincomycin was deleted by genetic engineering to obtain a lincomycin high-yield engineered strain, and the obtained strain was used to ferment and produce lincomycin; wherein the nucleotide sequence of the SLCG_3904 gene is shown in SEQ ID NO.

1.

2. The method for increasing lincomycin production by modifying the gene of Streptomyces lincomycin SLCG_3904 according to claim 1, characterized in that: The amino acid sequence encoded by the SLCG_3904 gene is shown in SEQ ID NO.

2.

3. The method for increasing lincomycin production by modifying the gene of Streptomyces lincomycin SLCG_3904 according to claim 1, characterized in that: The SLCG_3904 gene product negatively regulates lincomycin biosynthesis.

4. An application of the method according to any one of claims 1 to 3, characterized in that: The SLCG_3904 gene was knocked out in the industrial strain to obtain a high-yield mutant for lincomycin production.

5. The use according to claim 4, characterized in that: The industrial strain specifically selected is the high-yield strain LA219X.

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