Method for improving lincomycin yield by transforming streptomyces lincomycin SLCG-1587 gene and application

Abstract

The invention discloses a method for improving the yield of lincomycin by transforming a streptomyces lincomycin SLCG-1587 gene, in the streptomyces lincomycin, a GntR family transcriptional regulatory gene SLCG-1587 is knocked out through a genetic engineering way, the nucleotide sequence of the SLCG-1587 gene is shown as SEQ ID NO.1, a lincomycin high-yield engineering strain is obtained, and the lincomycin yield is improved. The yield of lincomycin produced by fermentation of the obtained strain can be greatly increased, and a new technical support is provided for increasing the yield of lincomycin 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-1587. Background technology:

[0002] Lincomycin is a lincosamide antibiotic that is widely used in various industries, especially in clinical practice. It has a good inhibitory effect on methoxy-resistant Staphylococcus aureus. With the increasing market demand, the level of industrial production needs to be improved accordingly. Microbial fermentation to produce lincomycin is currently the most promising method, and high-yield strains are the key to the industrial production of lincomycin. At present, the fermentation time of lincomycin is relatively long, the cell metabolism is complex during the fermentation process, and the hyphae are prone to agglomeration and aging, resulting in low lincomycin production. With the development of molecular biology, a large number of studies have been conducted on the synthesis of antibiotics at home and abroad. It was found that the production of lincomycin can be effectively increased by modifying the core genes of the lincomycin biosynthesis pathway. Therefore, it is necessary to explore the biosynthesis of lincomycin and its regulatory pathways, so as to carry out targeted transformation of lincomycin industrial strains.

[0003] Lincomycin biosynthesis is regulated by key transcriptional regulatory factors, including regulatory factors in gene clusters, pleiotropic regulatory factors, and global transcriptional regulatory factors, which play a vital role in the primary and secondary metabolism of Streptomyces lincomycin. Global regulatory factors can regulate morphological differentiation (hyphae growth, spore formation, pigment synthesis, etc.) and secondary metabolism. Studies on Streptomyces coelicolor and other Streptomyces have shown that many typical global regulatory factors are ubiquitous in Streptomyces and transmit various input signals to genes, including nutrient utilization, translation rate, developmental state, external pressure and other environmental information.

[0004] The GntR family transcriptional regulatory factors are the most widely distributed type of helix-turn-helix transcriptional regulatory factors in bacteria. These transcription factors regulate many different cellular processes of bacteria, such as motility, glucose metabolism, bacterial drug resistance, pathogenicity of pathogenic bacteria, etc. According to the different regulatory mechanisms, they are divided into two categories: positive transcriptional regulation (transcription activation) and negative transcriptional regulation (transcriptional repression). The vast majority of GntR family transcriptional regulatory factors are transcriptional repressors. The present invention screened the SLCG-1587 gene, which is highly homologous to the LcbR1 gene, in Streptomyces lincomycin through bioinformatics comparison analysis. Accordingly, a method for increasing the yield of lincomycin by modifying the SLCG-1587 gene of Streptomyces lincomycin was attempted. Summary of the invention:

[0005] 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-1587.

[0006] The present invention adopts the following technical solutions to solve the above technical problems:

[0007] A method for increasing the yield of lincomycin by modifying the SLCG-1587 gene of Streptomyces lincomycin, wherein the SLCG-1587 gene in Streptomyces lincomycin is deleted through genetic engineering to obtain a high-yield lincomycin strain, and the obtained strain is used to ferment and produce lincomycin; wherein the nucleotide sequence of the SLCG-1587 gene is shown in SEQ ID NO.1.

[0008] As one of the preferred embodiments of the present invention, the amino acid sequence encoded by the SLCG-1587 gene is shown in SEQ ID NO.2.

[0009] As one of the preferred embodiments of the present invention, the SLCG-1587 gene expression product negatively regulates lincomycin biosynthesis.

[0010] The invention discloses an application of a method for improving the yield of lincomycin by modifying the lincomycin Streptomyces lincomycin SLCG-1587 gene, wherein the SLCG-1587 gene is knocked out in an industrial strain of the lincomycin Streptomyces lincomycin to obtain a high-yield mutant strain, and the obtained strain is used for fermentation production of lincomycin.

[0011] The advantages of the present invention are:

[0012] In the present invention, the negative regulator of lincomycin biosynthesis SLCG-1587 was screened out, and the SLCG-1587 gene on the genome of Streptomyces lincomycin was knocked out by genetic engineering, so that a high-yield strain of lincomycin can be constructed, providing technical support for increasing the yield of lincomycin in industrial production.

[0013] The lincomycin production of the SLCG-1587 deletion mutant in Streptomyces lincomycin L-427 was increased, indicating that SLCG-1587 is a negative regulator involved in lincomycin biosynthesis. Description of the drawings:

[0014] Figure 1 This is a location information diagram of the SLCG-1587 gene and its surrounding adjacent genes on the genome in Example 1.

[0015] Figure 2 Shown is a schematic diagram of the construction of the SLCGL-1587 knockout plasmid pOJ260-neo-Δ1587.

[0016] Figure 3 is the PCR identification of the ΔSLCGL-1587 mutant.

[0017] Figure 4 is the dry weight of L-427 and ΔSLCG-1587 fermentations.

[0018] Figure 5 The figure shows the lincomycin production analysis of WT (L-427) and ΔSLCG-1587 fermentations. Specific implementation plan:

[0019] 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.

[0020] The strains and plasmids used in the following examples are shown in Table 1, and the synthesized primer sequences are shown in Table 2. The Escherichia coli used in the examples were cultured in liquid LB medium at 37°C or on solid LB plates supplemented with 2% agar. Streptomyces lincomyces was cultured in mycelial TSB medium at 30°C or on modified slant plates containing 2% agar.

[0021] Kanamycin used in the following examples was purchased from Sangon Biotech Co., Ltd. Peptone, yeast extract, and TSB were purchased from Oxoid. Glucose, agar, sodium chloride, and other biological reagents were purchased from a reagent company. General operation techniques for Escherichia coli and Streptomyces lincomycin were performed according to standard procedures. Primer synthesis was performed by Beijing Qingke Biotechnology Co., Ltd., and DNA sequencing was performed by Shanghai Jieli Biotechnology Co., Ltd.

[0022] Table 1 The present invention relates to strains and plasmids.

[0023]

[0024] Table 2 The present invention relates to primers

[0025]

[0026]

[0027] Example 1 SLCG-1587 gene related information

[0028] Based on the gene annotation information in the NCBI database, the location information of SLCG-1587 and its surrounding genes in the genome of Streptomyces lincomyces (CP022744.1) and the potential gene functions are as follows Figure 1 shown.

[0029] The nucleotide sequence of the SLCG1587 gene is shown in SEQ ID NO.1, with a total length of 705 bp; the encoded amino acid sequence is shown in SEQ ID NO.2, consisting of 234 amino acids.

[0030] TCAGAGGAGGGCTTCGCCCTCCCCCACACGCCGCCTGTAGACCTCGACGACCCGTTCCAGGGAGTCGCTCAGGTATACCGCGAGGAGCTTTTCGGCCTCCGTCTTGTCGCCCGCCTGCAACGCTCCGAGGATCTGCTGGTTGCGGGCCAGATACGGCTCGTGCAGGCGGCGGGGGTCGTCCACGACGTGGAAGGCGAGGCGGAGTTCGGCGAAGACACTGCGCATCAGTTCGTCGGTGCGGTCGCTGCCGGCCAGGGCGACGAGTTCGCGGTGGAAGTGGATGTTGGCCGTACCCAGCACCTTCCAGTCACCTTCGCGTACGGCCCGCTGTCCCTCGGCCACGGCCTCGGCGAGGCCGTCGAGGGCGTAGGGCGGCTCCCCGAGGTCCCGTACGACGGCGCACTCGACGAGGCCGCGGGTGCGGTAGATGTCCTCCACGTCCTCCACGGTCAGCACCCGGACGAAGACGCCCCGGTTCAGCTCGTGGACGAGCAGGCGTTCGTGGGTGAGCAGCCGGAACGCCTCGCGCAGCGTGTTGCGGGAGACGCCGAGCGCGCCCCCGATGCTGTCCTCCGAGAGCCGGGTCCCCGGCGGGAAGAAGCCCTCGGCGATGCGGCTCCTGAGGATGTCCGAGACCCGCTCGGCCGTGCTGGTGCGCCCCAGGAGGGCCCGGTCGTCGGCCAGTCCGCTCAGCTGTTCCGCCAT(SEQ ID NO.1)

[0031] MAEQLSGLADDRALLGRTSTAERVSDILRSRIAEGFFPPGTRLSEDSIGGALGVSRNTLREAFRLLTHERLLVHELNRGVFVRVLTVEDVEDIYRTRGLVECAVVRDLGEPPYALDGLA EAVAEGQRAVREGDWKVLGTANIHFHRELVALAGSDRTDELMRSVFAELRLAFHVVDDPRRLHEPYLARNQQILGALQAGDKTEAEKLLAVYLSDSLERVVEVYRRRVGEGEALL(SEQ ID NO.2)

[0032] Example 2 Construction of knockout plasmid pOJ260-neo-Δ1587

[0033] The SLCG-1587 gene knockout plasmid was constructed using the Escherichia coli-Streptomyces shuttle plasmid pOJ260-neo.

[0034] Primers were designed using pOJ260-neo plasmid and the genome of S. lincolnensis L-427 as templates (Table 1). 1587-UF / 1587-UR and 1587-DF / 1587-DR were used as primers to amplify homologous fragments of about 1.4 kb upstream and downstream of the SLCG-1587 gene by PCR.

[0035] Use the Bio-Tech Seamless Cloning Kit to connect the upstream homology arm 1587-U to the HindⅢ site of the pOJ260-neo vector backbone to obtain plasmid 1. Prepare the system according to 2X Seamless Cloning Mix 5μL, insert fragment, vector backbone 50ng, and ddH2O to fill to 10μL. After the system is prepared, gently pipette to mix, and place it in a 50℃ PCR instrument for reaction for 15min. After the 50℃ reaction is completed, it can be directly transformed or frozen in a -20℃ refrigerator.

[0036] Plasmid 1 was transformed into the competent DH5α E. coli host using the Bio-Tech Seamless Cloning Kit. PCR verification was performed using agarose gel electrophoresis, and plasmids with correct and clear colony PCR bands were selected for sequencing. Finally, the correctly sequenced plasmid and DH5α E. coli containing the pOJ260-neo-Δ1587U vector were cryopreserved, and the construction of the pOJ260-neo-Δ1587U vector was completed.

[0037] Use the Bio-Tech Seamless Cloning Kit to connect the downstream homology arm 1587-D to the EcoRI site of the pOJ260-neo-Δ1587U vector backbone to obtain plasmid 2. After the system is prepared, gently pipette to mix, and place it in a 50℃ PCR instrument for reaction for 15 minutes. After the 50℃ reaction is completed, it can be directly transformed or frozen in a -20℃ refrigerator.

[0038] Plasmid 2 was transformed into the competent E. coli host DH5α using the Bio-Tech Seamless Cloning Kit. PCR verification was performed using agarose gel electrophoresis, and plasmids with correct and clear colony PCR bands were selected for sequencing. Finally, the correctly sequenced plasmid and DH5α E. coli containing the pOJ260-neo-Δ1587 vector were cryopreserved, and the construction of the pOJ260-neo-Δ1587 vector was completed.

[0039] Example 3 Transformation of recombinant plasmid

[0040] 1) Transform the pOJ260-neo-Δ1587 vector into methylation-deficient Escherichia coli competent cell ET12567 (pUZ8002);

[0041] 2) Perform seed culture of wild-type Streptomyces lincomyces L-427, gently scrape spores from the plate with a sterile cotton swab, suspend in 1 mL of TSB, and heat shock in a 50°C water bath for 12 minutes. After cooling to room temperature, pre-germination culture was performed at 37°C, 220 rpm for 2-8 hours; the pre-germinated spores were centrifuged at 4000 rpm for 5 minutes, and the supernatant was discarded for later use.

[0042] 3) Inoculate the E. coli constructed in step 1) into 3 mL of LB medium (containing the corresponding resistance) containing 0.5‰, and culture at 37°C overnight; inoculate into 3 mL of LB medium (containing the corresponding resistance) containing 0.5‰ with an inoculum size of 3%, and culture at 37°C with shaking until OD 600 0.4-0.6. Centrifuge at 5000rpm for 5min, remove the supernatant and collect the cells. Add 1mL of LB medium without antibiotics to wash the cells thoroughly, centrifuge at 5000rpm for 5min, remove the supernatant, repeat this step once. Resuspend the cells in 200μL of LB liquid medium without resistance and set aside.

[0043] The mycelium of Streptomyces lincosus obtained in step 2) was mixed with the suspension of Escherichia coli obtained in step 3), and coated on a solid ISP4 plate containing 30mM magnesium chloride, and inverted in a 30°C incubator. After culturing for 20 hours, a covering liquid (1mL sterile water, 1μL nalidixic acid at a concentration of 50g / mL and 1μL kanamycin at a concentration of 50g / mL) was added, and the mixture was mixed by blowing and suction, and then covered on the plate. After the surface moisture of the culture medium was dried, the culture was inverted in a 30°C incubator for about 7 days to see the conjugants; the transformants were screened and amplified on the SMA solid plate.

[0044] Example 4 Obtaining SLCG-1587 knockout strain

[0045] Several single colonies were picked from the plates with successful conjugation and transferred, and PCR verification was performed using 1587-CF and 1587-CR as identification primers ( Figure 3 ), a genetically engineered strain with SLCG-1587 gene deletion was obtained, and a 1101bp band was obtained by PCR amplification of the genome of the SLCG-1587 deletion strain. After agarose gel electrophoresis verification, the correct fragment of the band was recovered and sent for sequencing and compared with the plasmid pOJ260-neo-Δ1587 to ensure that the strain was constructed correctly. The constructed strain was named ΔSLCG-1587.

[0046] Example 5 Fermentation process of genetically engineered strain (mutant strain) producing lincomycin and extraction and determination of metabolites

[0047] (1) Fermentation process and metabolite extraction of mutant strains and wild-type strains

[0048] Use a sterilized inoculation shovel to scoop 1 cm above the solid culture medium of Streptomyces lincomyces 2 The fresh bacterial mass was transferred into 30 mL of primary fermentation medium (250 mL shake flask) and cultured at 30°C, 220 rpm for 48 h. The bacterial liquid in the primary fermentation medium was transferred into 30 mL of fermentation medium (250 mL shake flask) at 10% inoculum and cultured at 30°C, 220 rpm for 168 h.

[0049] (2) Dry weight detection

[0050] 1. Use a precision balance to weigh the mass of a 2mL clean EP tube;

[0051] 2. Pipette 1 mL of fermentation liquid into a 2 mL EP tube, centrifuge at 12000 rpm for 5 min, and remove the supernatant;

[0052] 3. Add 1 mL of sterile water to wash the cells, centrifuge at 12000 rpm for 5 min, and remove the supernatant;

[0053] 4. Open the EP tube after collecting the bacteria and place it in an oven to dry until the mass no longer changes. Record the data and subtract the mass of the empty tube to get the measured dry weight.

[0054] The dry weight of lincomycin-producing Streptomyces lincomycin L-427 and SLCG-1587 knockout strains was as follows Figure 4 shown.

[0055] (3) Determination of the potency of the metabolite lincomycin

[0056] Take 1mL of the fermentation liquid after the fermentation is completed, add 1mL of 0.05mol / L NaOH solution to dilute, add 2mL of n-butanol after dilution, mix well and centrifuge at 3000rpm for 3min; take 1mL of the above extract, add 2mL of 0.1mol / L hydrochloric acid, mix well and let stand for stratification; take 1mL of the above back extract, add excess 0.2mol / L palladium chloride solution to make the volume 10mL, mix well and place in a 30℃ water bath to react for 20-30min, take out and cool to room temperature, and measure OD at a wavelength of 380nm 380 The standard curve is based on the lincomycin concentration as the ordinate, and the OD 380 As the horizontal axis, the linear regression equation is obtained.

[0057] Analysis of lincomycin production in lincomycin-producing Streptomyces lincomycin L-427 and SLCG-1587 knockout strains Figure 5 shown.

[0058] (4) Measurement results

[0059] The results of the embodiment show that SLCG-1587 does not affect the production of spores. The dry weight of ΔSLCG-1587 gradually increases and reaches the highest at 96h, and then gradually decreases. Figure 4 The results of lincomycin production analysis are as follows: Figure 5 As shown, compared with the wild-type strain L-427, knocking out the SLCG-1587 gene during the fermentation process significantly increased the production of lincomycin. At the end of the fermentation, the lincomycin production of the wild-type strain L-427 was 439.2912 μg / mL, and the lincomycin production of ΔSLCG-1587 was 975.7224 μg / mL, which was about 2.22 times that of the wild-type.

Claims

1. A method for increasing the yield of lincomycin by modifying the gene of Streptomyces lincomycin SLCG-1587, characterized in that: By knocking out the GntR family transcriptional regulatory gene SLCG-1587 through genetic engineering, a high-yield engineered strain of lincomycin is obtained, and the yield of lincomycin produced by fermentation with the obtained strain can be greatly increased.

2. The method for increasing lincomycin production by modifying the gene of Streptomyces lincomycin SLCG-1587 according to claim 1, characterized in that: The amino acid sequence of the SLCG-1587 gene is shown in SEQ ID NO.

2.

3. The method for increasing lincomycin production by modifying the gene of Streptomyces lincomycin SLCG-1587 according to claim 1.

4. An application of the method for increasing lincomycin production by genetically modifying Streptomyces lincomycin SLCG-1587 as claimed in any one of claims 1 to 3, characterized in that: The SLCG-1587 gene was knocked out in the lincomycin industrial strain to obtain a high-yield mutant for the production of lincomycin.

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