Method for improving lincomycin yield by transforming streptomyces lincomycin SLCG6736 gene and application

Abstract

The invention relates to the technical field of genetic engineering, and provides a method for improving the yield of lincomycin by modifying a streptomyces lincomycin SLCG6736 gene, the SLCG6736 gene in the streptomyces lincomycin is deleted through a genetic engineering way to obtain a lincomycin high-yield engineering strain, and the obtained strain is used for fermentation production of lincomycin. Wherein the nucleotide sequence of the SLCG6736 gene is as shown in SEQ ID NO. 1. The invention further provides application of the method. The lincomycin biosynthesis negative regulator SLCG6736 has the advantages that the lincomycin biosynthesis negative regulator SLCG6736 is screened in the research, the SLCG6736 gene is knocked out 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 of increasing the yield of lincomycin by transforming the gene of Streptomyces lincomycin SLCG_6736. Background Art

[0002] Microorganisms can produce secondary metabolites with diverse structures and rich varieties, and are a treasure trove for the research and development of natural medicines. Microbial natural medicines used in clinical practice are mainly derived from actinomycetes, and half of the antibacterial drugs are derived from actinomycetes. Most of these substances have antibacterial, antifungal, antitumor, immunosuppressive, and insecticidal activities, and are widely used in the pharmaceutical, agricultural, and food industries.

[0003] Streptomyces lincomycin is a major actinomycete in the industrial fermentation production of lincomycin. The active component A is used to treat infections caused by Gram-positive bacteria such as Staphylococcus aureus and Streptococcus pneumoniae, and some anaerobic Gram-negative bacteria. Lincomycin binds to the central ring of the 23S rRNA of the bacterial 50S ribosome subunit, inhibiting the localization of tRNA in the peptidyl transferase center and thus affecting protein biosynthesis. Lincomycin has strong penetration into tissues and cells and is widely used in clinical practice.

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

[0005] NmrA (Nitrogen metabolite repression A) is a repressor of nitrogen metabolism regulation and is involved in regulating the activity of AreA transcription factor. Glutamine, as a signal molecule of intracellular nitrogen levels, can be recognized by NmrA protein. In an environment containing a preferred nitrogen source, NmrA is activated and binds to the AreA transcription factor to change the conformation of the AreA protein, causing the AreA protein to lose the ability to bind to DNA, and ultimately inhibiting the expression of genes related to secondary nitrogen source metabolism. When there is no preferred nitrogen source such as ammonium nitrogen and glutamine in the environment, NmrA separates from AreA, and active AreA binds to the promoter region of genes related to secondary nitrogen source metabolism, thereby activating the transcription of genes related to secondary nitrogen source metabolism. 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. Based on this, the present invention screened the SLCG_6736 gene highly homologous to the nmrA gene in Streptomyces lincomycin through bioinformatics comparison analysis, and attempted to transform the SLCG_6736 gene of Streptomyces lincomycin to increase the lincomycin yield. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a method and application for increasing the yield of lincomycin by transforming the gene of Streptomyces lincomycin SLCG_6736.

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

[0008] A method for improving the yield of lincomycin by modifying the SLCG_6736 gene of Streptomyces lincomycin, wherein the SLCG_6736 gene 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_6736 gene is shown in SEQ ID NO.1.

[0009] As one of the preferred embodiments of the present invention, the amino acid sequence encoded by the SLCG_6736 gene is shown in SEQ ID NO.2.

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

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

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

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

[0014] In the present invention, the negative regulator of lincomycin biosynthesis SLCG_6736 was screened out, and the SLCG_6736 gene was knocked out by genetic engineering to obtain a lincomycin high-yield strain, providing technical support for increasing the fermentation yield of lincomycin in industrial production.

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

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

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

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

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

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

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

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

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

[0024] 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, “***”: P < 0.001, “ns”: no significant difference);

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

[0026] The following is a detailed description of an embodiment of the present invention. This embodiment is implemented 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.

[0027] The strains and plasmids used in the following examples are shown in Table 1, and the synthesized primer sequences are shown in Table 2. Among them, Streptomyces lincone LCGL is 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.

[0028] Escherichia coli used in the following examples were cultured in liquid LB medium at 37°C or on solid LB medium supplemented with 1.25% agar. Streptomyces lincomycin was cultured in tryptone soy broth (TSBY) medium at 30°C or on modified Gould's 1 (MGM) plates containing 1.8% agar.

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

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

[0031]

[0032]

[0033] Table 2 The present invention relates to primers

[0034]

[0035]

[0036]

[0037] Example 1

[0038] SLCG_6736 gene related information:

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

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

[0041] Example 2

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

[0043] Using the LCGL genome as a template, 6736-P1, 6736-P2, 6736-P3, and 6736-P4 were used as primers to amplify 1.5 kb of the upstream and downstream homology arms of SLCG_6736, respectively, and recovered. At 37°C, the upstream and downstream homology arms were digested and recovered and quantified using HindIII / XbaI and KpnI / EcoRI, respectively. While digesting the homology arms, the pKC1139 plasmid was digested with EcoRI / HindIII and 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 to grow a single clone. Finally, a single clone was picked and transferred to LB liquid culture medium containing Apr resistance for expansion culture, and then transferred to 5 mL of LB medium containing Apr resistance and cultured for 12 hours to extract the pKC1139-Δ6736 plasmid.

[0044] The obtained pKC1139-Δ6736 plasmid was transformed into the protoplasts of LCGL by PEG3350, and the construction of SLCG_6736 knockout strain was completed by homologous large fragment recombination technology. The specific operation is as follows:

[0045] Take 3000ng of pKC1139-Δ6736 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 about 20h. When a membranous bacteria grows on its surface, add Apr antibiotics for covering screening. After 4d, a single clone with a black bottom grows on the plate, which is enriched on an industrial plate containing Apr and cultured at 30℃ for 3d. Subsequently, pick the enriched spores, dilute them 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 are spread on the industrial plate without resistance. Select a single clone that cannot grow on the Apr plate but can grow on the non-antibiotic plate and culture it until it produces spores. Then scrape a certain amount of spores into sterile water, boil them at high temperature for 15 minutes, and then perform PCR identification using 6736-P5 and 6736-P6 as primers. Figure 3 , and obtain the correct knockout strain.

[0046] Example 3

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

[0048] Using the LCGL genome as a template, 6736-P7 and 6736-P8 as primers, the SLCG_6736 gene fragment with NdeI and XbaI restriction sites at both ends was amplified and recovered. The SLCG_6736 fragment and the pIB139 plasmid were double-digested with NdeI and XbaI and recovered. At 21°C, T4 ligase was added to connect the SLCG_6736 and pIB139 plasmids and transformed into Escherichia coli DH5α. Spread on an LB plate with Apr resistance, and a single clone will grow at 37°C for about 12 hours. Pick a single clone and transfer it to liquid LB culture medium for 5 hours of culture, and then perform bacterial liquid PCR identification. The correctly identified bacteria will be sent for sequencing, and after sequencing is correct, the seeds will be preserved to obtain the pIB139-6736 plasmid.

[0049] The pIB139-6736 plasmid was introduced into LCGL and ΔSLCG_6736 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_6736 / pIB139-6736 and LCGL / pIB139-6736 strains were obtained.

[0050] Example 4

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

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

[0053] Example 5

[0054] HPLC detection of fermentation products of Streptomyces lincomyces:

[0055] After culturing Streptomyces lincosus on the slant medium for 7 days, 1 cm 2 The spore mass was inoculated into the seed culture medium and cultured at 30°C and 240 rpm for 48 hours. Then, it was transferred to the fermentation medium and cultured at 30°C and 240 rpm for 7 days. Then, 2 mL of bacterial solution was centrifuged at 12000 rpm for 10 minutes, and 200 μL of supernatant was mixed with 800 μL of anhydrous ethanol and centrifuged at 12000 rpm for 10 minutes. Finally, the supernatant was injected into the detection bottle through an organic filter membrane for yield detection.

[0056] Example 6

[0057] Streptomyces lincomyces mycelium biomass detection:

[0058] LCGL and ΔSLCGL_6736 were inoculated into 5 mL of liquid TSBY with the same inoculation amount, and cultured at 30°C for 48 h. Take an equal amount of fresh bacterial solution of LCGL and ΔSLCGL_6736 in YMG medium, culture at 30°C and 240 rpm for 7 days, and take out 1 mL of fresh bacterial solution every 24 hours and store it in a -20°C refrigerator. After the fermentation is completed, centrifuge the 7d sample at 12000 rpm for 10 min and discard the supernatant. Add 1 mL of anhydrous ethanol to wash the bacteria, centrifuge at 12000 rpm for 10 min, and discard the supernatant. Place the wet bacteria in a 65°C oven, dry for 2 days, weigh, and record the mass of the bacteria. The dry weight of the bacteria of ΔSLCGL_6736 and LCGL is plotted according to the growth time.

[0059] Example 7

[0060] Transcription analysis of related genes in ΔSLCG_6736:

[0061] The LCGL and ΔSLCG_6736 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.

[0062] Example 8

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

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

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

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

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

[0068] 3. SLCG_6736 gene complementation and overexpression

[0069] In order to further verify that the increase in lincomycin production in ΔSLCGL_6736 was caused by the deletion of the SLCG_6736 gene, the SLCG_6736 gene expression vector pIB139-6736 was introduced into the protoplasts of LCGL and ΔSLCGL_6736, and the complemented strain ΔSLCGL_6736 / pIB139-6736 and the overexpression strain LCGL / pIB139-6736 were obtained by PCR identification. The LCGL and ΔSLCGL_6736 series of strains were fermented in shake flasks, and the HPLC test results showed that the lincomycin production in ΔSLCGL_6736 was 16.6% higher than that in LCGL; the lincomycin production in the complemented strain ΔSLCGL_6736 / pIB139-6736 was restored to the level of lincomycin in LCGL; the lincomycin production in LCGL / pIB139-6736 was reduced by 16.2% compared with LCGL ( Figure 8 ). These results indicate that SLCG_6736 negatively regulates the biosynthesis of lincomycin.

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

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

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

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

[0074] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for increasing the yield of lincomycin by modifying the gene of Streptomyces lincomycin SLCG_6736, characterized in that: The SLCG_6736 gene in Streptomyces lincomycin is deleted by 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_6736 gene is shown in SEQ ID NO.

1.

2. The method for increasing lincomycin production by modifying the gene of Streptomyces lincomycin SLCG_6736 according to claim 1, characterized in that: The SLCG_6736 gene product negatively regulates lincomycin biosynthesis.

3. An application of the method according to claim 1 or 2, characterized in that: The SLCG_6736 gene was knocked out in the industrial strain to obtain a high-yield mutant strain that can be used for lincomycin production.

4. The use according to claim 3, characterized in that: The industrial strain specifically selected is the high-yield strain LA219X.

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