Method and application for increasing lincomycin yield by modifying Streptomyces lincolnensis SLCG_3904 gene

Abstract

The present invention provides a method for increasing the yield of lincomycin by modifying the SLCG_3904 gene of Streptomyces lincolnensis. Through genetic engineering, the GlnR family transcriptional regulatory gene SLCG_3904 in Streptomyces lincolnensis is deleted to obtain a high-yield engineering strain of lincomycin, and the obtained strain is used for fermentative production of lincomycin. Among them, the nucleotide sequence of the SLCG_3904 gene is as shown in SEQ ID NO.1. The present invention also provides the application of the above method. The advantages of the present invention are as follows: In the research of the present invention, the negative regulator SLCG_3904 (GlnR family) of lincomycin biosynthesis is screened. By deleting the SLCG_3904 gene on the chromosome of Streptomyces lincolnensis through genetic engineering, a high-yield strain of lincomycin can be obtained, providing technical support for improving the fermentation yield of lincomycin in industrial production.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly relates to a method and application for improving the production of lincomycin by modifying the SLCG_3904 gene of Streptomyces lincolnensis. Background Art

[0002] Streptomyces is an important source of secondary metabolites and can produce secondary metabolites with various biological activities, such as antifungal, antiviral, antitumor, and antihypertensive compounds, etc. The most important ones are antibiotics and immunosuppressants.

[0003] Lincomycin, a lincosamide antibiotic mainly produced by Streptomyces lincolnensis, and its derivative clindamycin are both very important antibiotics in clinical practice. They act on Gram-positive bacteria and some Gram-negative bacteria, and their action site is the peptidyl transferase center of the bacterial ribosome. By binding to the central loop of the 23S rRNA in the 50S subunit, they hinder the elongation of the peptide chain, inhibit the synthesis of proteins in bacteria, and thus affect the growth of bacteria. Lincomycin is commonly used to treat bone and joint infections, and can also prevent postoperative intraperitoneal infections, oral infections, skin and mucosal infections, etc. It has high efficacy, low toxicity, and is not prone to cross-resistance, and is a class of antibiotics with broad application prospects.

[0004] With the rapid progress of whole-genome sequencing technology, more and more genomic sequences of Streptomyces lincolnensis have been determined and analyzed, but it is found that most of its biosynthetic gene clusters are in a silent state or have weak secondary metabolite synthesis ability. These gene clusters need to be screened to obtain 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 high-yield strains of lincomycin by genetically engineering to change genes directionally, for the production of lincomycin or intermediate products.

[0005] GlnR (Glutamine Synthetase Regulatory Protein) is widely present in bacteria and archaea. It is a global transcriptional regulatory protein in bacteria and plays a key role in many bacteria, mainly participating in the regulation of nitrogen metabolism. Research has found that GlnR can adapt to environmental changes by directly regulating multiple genes related to nitrogen metabolism, such as urea metabolism and nitrate reduction. The global regulatory factor GlnR 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 the bacteria. In addition, GlnR also increases the accumulation of glutamate in cells by promoting the transcriptional level of related genes in the nitrate assimilation pathway. Glutamate can form the precursor tyrosine for lincomycin synthesis through transamination. At the same time, research has found that when Streptomyces lincolnensis performs antibiotic biosynthesis, adding a certain amount of nitrate will affect the primary metabolism of the antibiotic and further affect the process of antibiotic production.

[0006] Accordingly, in the present invention, the SLCG_3904 gene of the GlnR family was screened in Streptomyces lincolnensis, and an attempt was made to modify the SLCG_3904 gene of Streptomyces lincolnensis 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 modifying the SLCG_3904 gene of Streptomyces lincolnensis.

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

[0009] A method for increasing the yield of lincomycin by modifying the SLCG_3904 gene of Streptomyces lincolnensis, which is to delete the transcriptional regulatory gene SLCG_3904 of the GlnR family in Streptomyces lincolnensis through genetic engineering to obtain a high-yield engineering strain of lincomycin, and use the obtained strain to ferment and produce lincomycin; wherein, the nucleotide sequence of the SLCG_3904 gene is as 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 as shown in SEQ ID NO.2.

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

[0012] An application of the above method: knocking out the SLCG_3904 gene in industrial strains to obtain high-yield mutant strains for the production of lincomycin.

[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 with the prior art are as follows:

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

[0016] Specifically, when the SLCG_3904 gene was knocked out in Streptomyces lincolnensis LCGL, the lincomycin yield increased by 18.2%, indicating that SLCG_3904 is a negative regulatory factor involved in lincomycin biosynthesis. Using the high-yield strain LA219X as the starting strain and knocking out the SLCG_3904 gene on its chromosome, the lincomycin yield increased by 12.8%, indicating that the technology of knocking out SLCG_3904 to increase the lincomycin yield is also applicable to industrial high-yield strains. Brief Description of the Drawings

[0017] Figure 1 It is a map of the position information of the SLCG_3904 gene and its neighboring genes on the chromosome;

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

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

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

[0021] Figure 5PCR identification results of the high-yield lincomycin strain ΔSLA219X_3904 (in the figure, "M": 5000bp DNA Marker; "+", pKC1139-Δ3904 plasmid; "-", LCGL; "1", ΔSLA219X_3904);

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

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

[0024] Figure 8 Analysis of lincomycin production of the 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] Figure 9 Analysis of the transcriptional levels of genes related to the lincomycin biosynthetic gene cluster (in the figure, "*": P < 0.1, "**": P < 0.01, "ns": no significant difference);

[0026] Figure 10 Analysis of lincomycin production in the high-yield lincomycin strains LA219X and ΔSLA219X_3904 (in the figure, "***": P < 0.001). Detailed implementation manners

[0027] The following details the embodiments of the present invention. These embodiments are implemented on the premise of the technical solution of the present invention, and provide detailed implementation manners and specific operation processes. However, the protection scope of the present invention is not limited to the following embodiments.

[0028] The strains and plasmids used in the following examples are shown in Table 1, and the sequences of the synthesized primers are shown in Table 2. Among them, Streptomyces lincolnensis LCGL is a strain obtained by improving on the basis of Streptomyces lincolnensis LC-G (GenBank: CP022744.1). Specifically, the SLCG_7011 gene (GenBank: AXG58166.1) of Streptomyces lincolnensis LC-G was replaced with 4×attBΦC31; the 4×attBΦC31 is a 240-bp base containing 4 attBΦC31 site sequences, and the base sequence is as shown in SEQ ID NO.3.

[0029] The Escherichia coli used in the following examples was cultured in liquid LB medium at 37 °C or on solid LB plates supplemented with 1.25% agar. Streptomyces lincolnensis was cultured in tryptone soy broth (TSBY) medium at 30 °C or on modified Czapek No. 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 all purchased from reagent companies. The general operating techniques for Escherichia coli and Streptomyces lincolnensis were in accordance with standard operations. The synthesis of primers and DNA sequencing were completed by Sangon Biotech (Shanghai) Co., Ltd.

[0031] Table 1 Strains and plasmids involved in the present invention

[0032]

[0033]

[0034] Table 2 Primers involved in the present invention

[0035]

[0036]

[0037]

[0038] Example 1

[0039] Information related to the SLCG_3904 gene:

[0040] The positions of the SLCG_3904 gene and its neighboring genes on the chromosome are shown 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 size of the protein monomer is approximately 28 KDa.

[0042] Example 2

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

[0044] Using the LCGL genome as a template and 3904-P1, 3904-P2, 3904-P3, 3904-P4 as primers, the upstream and downstream homologous arms of SLCG_3904, each 1.5 kb in length, were amplified separately and recovered. At 37 °C, XbaI / EcoRI and XbaI / HindIII were used to digest and recover the upstream and downstream homologous arms respectively and quantify them. While digesting the homologous arms, the pKC1139 plasmid was double-digested with EcoRI / HindIII, recovered and quantified. According to the quantification results, the digested plasmid, upstream homologous arm, and downstream homologous arm were mixed at a ratio of 1:10:10. At 22 °C, T4 ligase was added for ligation. Then, all the ligation products were transformed into Escherichia coli DH5α, and at 37 °C, they were diluted and spread on an LB plate resistant to apramycin (Apr) and cultured for about 12 h until monoclonal colonies grew. Finally, monoclonal colonies were picked into an LB liquid medium containing Apr resistance for amplification culture, and then transferred to 5 mL of LB medium containing Apr resistance and cultured for 12 h to extract the pKC1139-Δ3904 plasmid.

[0045] The obtained pKC1139-Δ3904 plasmid was transformed into the protoplasts of LCGL via PEG3350-mediated transformation, and the construction of the SLCG_3904 knockout strain was completed using the homologous large fragment recombination technique. The specific operation is as follows:

[0046] Mix 4000 ng of pKC1139-Δ3904 with 50 μL of LCGL protoplasts, then add 200 μL of PEG3350 and let it stand for 5 min. Dilute the mixture and spread it on the R5 plate, and culture it at 30 °C for 20 h; when membranous bacteria grow on its surface, add Apr antibiotic for covering and screening. After about 4 days, monoclonal colonies with black bottoms grow on the plate. Enrich them on the industrial plate containing Apr and culture them at 30 °C for about 3 days. Subsequently, pick the enriched spores, dilute and spread them on the antibiotic-free R5 plate, and culture them at a constant temperature of 37 °C for 3 days for plasmid loss. Finally, spread some monoclonal colonies on the R5 plate on the Apr industrial plate, and spread the remaining monoclonal colonies on the antibiotic-free industrial plate. Select the monoclonal colonies that cannot grow on the Apr plate but can grow on the antibiotic-free 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 15 min. Then use 3904-P5 and 3904-P6 as primers for PCR identification. As Figure 3 shown, the correct knockout strain ΔSLCGL_3904 is obtained.

[0047] Example 3

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

[0049] Using the LCGL genome as a template, amplify the SLCG_3904 gene fragment with NdeI and XbaI restriction sites at both ends using 3904-P7 and 3904-P8 as primers and recover it. Use NdeI and XbaI to double-digest the SLCG_3904 fragment and the pIB139 plasmid and recover them. At 22 °C, add T4 ligase to ligate SLCG_3904 and the pIB139 plasmid, and transform them into Escherichia coli DH5α. Spread them on the LB plate with Apr resistance and culture them at 37 °C for about 12 h until monoclonal colonies grow. Pick the monoclonal colonies and transfer them to the liquid LB medium for culture for 5 h, and then perform bacterial liquid PCR identification. Send the correctly identified bacteria for sequencing. After the sequencing is correct, preserve the strain to obtain the pIB139-3904 plasmid.

[0050] Transform the pIB139-3904 plasmid into LCGL and ΔSLCG_3904 protoplasts respectively by protoplast transformation. After 20 h of transformation, cover the medium with apramycin aqueous solution and culture it until transformants grow. Pick the transformants and spread them on the industrial plate with Apr resistance. Wait for the transformants resistant to Apr to grow, and use the primers Apr-F / R for verification. As Figure 4 shown, the correct ΔSLCGL_3904 / pIB139-3904 and LCGL / pIB139-3904 strains are obtained.

[0051] Example 4

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

[0053] The plasmid pKC1139-Δ3904 was transformed into the protoplasts of LA219X mediated by PEG3350 to construct the strain ΔSLA219X_3904. The construction and screening method refer to Example 3, and the PCR identification results are as Figure 5 .

[0054] Example 5

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

[0056] After Streptomyces lincolnensis was cultured on the slant medium for 7 days, a 1 cm 2 spore block was inoculated into the seed medium. After shaking culture at 30 °C and 240 rpm for 48 h, it was transferred to the fermentation medium and shaken culture at 30 °C and 240 rpm for 7 days. Then, 2 mL of the bacterial liquid was centrifuged at 12000 rpm for 10 min, and then 200 μL of the supernatant was mixed with 800 μL of absolute ethanol and centrifuged at 12000 rpm for 10 min. Finally, the supernatant was injected into the detection bottle through an organic filter membrane for yield detection.

[0057] Example 6

[0058] Detection of mycelial biomass of Streptomyces lincolnensis:

[0059] LCGL and ΔSLCGL_3904 were inoculated into 5 mL of liquid TSBY at the same inoculum amount and cultured on a shaker at 30 °C for 48 h. Equal amounts of fresh bacterial liquid of LCGL and ΔSLCGL_3904 were taken and cultured in YMG medium at 30 °C and 240 rpm for 7 days; during this period, 1 mL of fresh bacterial liquid was taken every 24 h and stored in a -20 °C refrigerator. After fermentation was completed, the samples of 7 days were centrifuged at 12000 rpm for 10 min, and the supernatant was discarded. 1 mL of absolute ethanol was added to wash the bacterial cells, and centrifuged at 12000 rpm for 10 min, and the supernatant was discarded. The wet bacterial cells were placed in an oven at 65 °C for 2 days, weighed, and the mass of the bacterial cells was recorded. The dry weights of the bacterial cells of ΔSLCGL_3904 and LCGL were used to plot the mycelial biomass curve according to the growth time.

[0060] Example 7

[0061] Transcription analysis of related genes in ΔSLCG_3904:

[0062] The bacterial liquid of LCGL and ΔSLCG_3904 at 24 h was collected, and the required RNA was obtained using the TransGen RNA extraction kit. After reverse transcription into cDNA, it was detected on a real-time fluorescence quantitative PCR instrument.

[0063] Example 8

[0064] Result analysis of the above embodiments:

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

[0066] The SLCG_3904 gene deletion mutant has been confirmed by PCR identification ( Figure 3 ). After ΔSLCGL_3904 was fermented in the fermentation medium for 168 h, the lincomycin yield was detected by HPLC. It was found that the yield of ΔSLCGL_3904 was about 18.2% higher than that of LCGL ( Figure 6 ). Based on the above results, it was confirmed that SLCG_3904 is a negative regulatory gene for lincomycin biosynthesis in Streptomyces lincolnensis. Inactivating the SLCG_3904 gene in Streptomyces lincolnensis through genetic engineering can increase the yield of lincomycin.

[0067] 2. Effects of SLCG_3904 gene deletion on spore morphological differentiation and cell growth

[0068] To determine whether the SLCG_3904 gene regulates spore formation of the cells, LCGL and ΔSLCGL_3904 were simultaneously spread on MGM plates and cultured at 30 °C for 7 d. The spore growth of the strains was observed daily. The results showed that there was no obvious difference in the spore morphology of ΔSLCGL_3904 compared with LCGL ( Figure 7 A), indicating that the deletion of the SLCG_3904 gene does not affect spore formation. At the same time, the dry cell weights of LCGL and ΔSLCGL_3904 were measured, and the corresponding change curves were drawn. The results showed that there was little difference in the biomass of ΔSLCGL_3904 compared with LCGL ( Figure 7 B), suggesting that the deletion of the SLCG_3904 gene does not affect the primary metabolism of the cells.

[0069] 3. Complementation and overexpression of the SLCG_3904 gene

[0070] To further verify that the increase in lincomycin production in ΔSLCGL_3904 is 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. Complemented strains ΔSLCGL_3904 / pIB139-3904 and overexpressing strains LCGL / pIB139-3904 were obtained through PCR identification. The LCGL and ΔSLCGL_3904 strains were subjected to shake flask fermentation. The HPLC detection 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 returned 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 indicate that SLCG_3904 negatively regulates lincomycin production.

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

[0072] The qRT-PCR data showed that compared with LCGL, the transcriptional levels of lmbA, lmbC, lmbD, lmbV, lmbZ, lmbJ, lmbK, lmrA, lmrB, lmrC, and lmbU in the lincomycin biosynthetic gene cluster in ΔSLCGL_3904 were up-regulated to varying degrees( Figure 9 ), indicating that SLCG_3904 negatively regulates the transcriptional levels of genes in the cluster to control lincomycin biosynthesis.

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

[0074] The ΔSLA219X_3904 strain and the high-yield strain LA219X were plated and activated, and then were respectively inoculated into shake flasks of industrial seed medium and cultured at 30 °C and 220 rpm for 48 h. They were transferred to the fermentation medium and cultured for another 168 h. After fermentation, extraction and concentration were carried out and analyzed by HPLC. Compared with LA219X, the lincomycin production of ΔSLA219X_3904 increased by 12.8%( Figure 10 ), indicating that the SLCG_3904 gene in the high-yield strain LA219X also participates in controlling lincomycin production.

[0075] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for increasing the yield of lincomycin by modifying Streptomyces lincolnensis SLCG_3904 is characterized in that By genetic engineering means, the GlnR family transcriptional regulatory gene in Streptomyces lincolnensis is SLCG_3904 deleted to obtain a strain with increased lincomycin production, and the obtained strain is used for fermentative production of lincomycin; wherein, SLCG_3904 the nucleotide sequence of the gene is as shown in SEQ ID NO.

1.

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

2.

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

4. Use of a strain with increased lincomycin production, characterized in that, The strain with increased lincomycin production is obtained by the method described in any one of claims 1 to 3; knocking out SLCG_3904 the gene to obtain the strain with increased lincomycin production, which is used for lincomycin production.

5. The application according to claim 4, wherein The specific industrial strain selected is strain LA219X.

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