Method for improving lincomycin yield by transforming streptomyces lincomycin SLCG2790 gene and application

Abstract

The invention provides a method for improving lincomycin yield by transforming a streptomyces lincomycin SLCG2790 gene, which comprises the following steps: knocking out the lincomycin SLCG2790 gene through a genetic engineering way to obtain a lincomycin high-yield strain, and fermenting the obtained strain to produce lincomycin A; wherein the nucleotide sequence of the SLCG2790 gene is as shown in SEQ ID NO. 1. The invention further provides application of the method. According to the invention, the SLCG2790 gene on the genome of the streptomyces lincomycin is knocked out through a genetic engineering way, so that the bacterium concentration of the streptomyces lincomycin L-427 on a fermentation tank can be improved, the yield of lincomycin A is improved, and a technical support is provided for improving 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_2790. Background Art

[0002] The morphological differentiation and secondary metabolism of Streptomyces are carried out synchronously and coupled to each other, and regulatory genes play an important role in this process. Regulatory factors not only affect the morphological differentiation of Streptomyces, but also regulate the secondary metabolism of Streptomyces. These secondary metabolic pathways are interconnected to form a complex metabolic network, and there are superposition and cascade effects between each network. At present, the research on regulatory factors related to these important metabolic pathways has established a metabolic regulatory network of Streptomyces. Changes in a single regulatory factor in the network will have an impact on the entire metabolism of Streptomyces. Finding and studying the nodes of the metabolic regulatory network will provide deep theoretical support for analyzing Streptomyces and transforming and utilizing them.

[0003] Streptomyces lincomycin is an actinomycete and the main producer of lincosamide antibiotic lincomycin. Lincomycin has good therapeutic effects on the treatment of infections caused by Gram-positive bacteria and some Gram-negative bacteria and is widely used in clinical practice. Lincomycin can also be used as a raw material to further chemically synthesize clindamycin, which has a high economic added value. my country is a major producer and exporter of lincomycin, but the industrial fermentation yield of lincomycin in my country is 8g / L, which is still behind the advanced foreign level of 10g / L. Directed modification through genetic engineering methods to increase lincomycin production is one of the commonly used ways to obtain antibiotic high-yield strains. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a method and application for improving the yield of lincomycin by transforming Streptomyces lincomycin SLCG_2790.

[0005] The present invention adopts the following technical solutions to solve the above technical problems:

[0006] A method for improving the yield of lincomycin by modifying the SLCG_2790 gene of Streptomyces lincomycin, wherein the SLCG_2790 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_2790 gene is shown in SEQ ID NO.1.

[0007] As one of the preferred embodiments of the present invention, the amino acid sequence encoded by the SLCG_2790 gene is shown in SEQ ID NO.2.

[0008] As one of the preferred embodiments of the present invention, the SLCG_2790 gene expression product negatively regulates lincomycin biosynthesis.

[0009] An application of the method for increasing the lincomycin yield by modifying the lincomycin SLCG_2790 gene is to knock out the SLCG_2790 gene in an industrial strain of lincomycin to obtain a high-yield mutant for lincomycin A production.

[0010] As one of the preferred embodiments of the present invention, the industrial strain of Streptomyces lincomyces is specifically selected as Streptomyces lincomyces L-427, which is isolated from soil in this laboratory and obtained through multiple rounds of mutagenesis and screening. It has industrial application potential and is preserved in this laboratory.

[0011] The advantages of the present invention compared to the prior art are:

[0012] In the present invention, the negative regulator of lincomycin biosynthesis SLCG_2790 was screened, the SLCG_2790 gene on the genome of Streptomyces lincomycin was knocked out by genetic engineering, and a lincomycin high-yield strain was constructed, providing technical support for increasing lincomycin yield in industrial production.

[0013] Specifically, by knocking out the SLCG_2790 gene in Streptomyces lincomycin L-427, the lincomycin A production can be increased by 37.7%; and at the 5L fermentation tank level, the lincomycin A production increased by 39.6%; the above indicates that the expression product of the SLCG_2790 gene negatively regulates the synthesis of lincomycin A. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the location information map of the SLCG_2790 gene and its neighboring genes on the genome;

[0015] Figure 2 is a schematic diagram of the construction of the L-427Δ2790 mutant;

[0016] Figure 3 is the result of PCR identification of the genome of L-427Δ2790;

[0017] Figure 4 It is a graph of lincomycin A production at the shake flask level of Streptomyces lincomycinus L-427 and L-427Δ2790 mutant;

[0018] Figure 5 It is a graph showing the change in horizontal wet volume of a 5L fermenter of Streptomyces lincomyces L-427 and L-427Δ2790 mutant;

[0019] Figure 6 This is the curve of lincomycin A production change in 5L fermentation tank of Streptomyces lincomycin L-427 and L-427Δ2790 mutant; DETAILED DESCRIPTION

[0020] 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.

[0021] The strains and plasmids used in the following examples are shown in Table 1, and the synthetic primer sequences are shown in Table 2.

[0022] The Escherichia coli used in the following examples were cultured in a liquid LB medium at 30°C or on a solid LB plate supplemented with 1.5% agar. The Streptomyces lincomyces was cultured in a TSB liquid medium at 30°C or on a solid slant medium plate.

[0023] TES and apramycin used in the following examples were purchased from Sinopharm Chemical Reagent Co., Ltd. TSB was purchased from Oxoid. Chloramphenicol, kanamycin, and nalidixic acid were purchased from Sangon Biotech Co., Ltd. Agar and other chemicals were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. ISP4 medium was purchased from BD Biosciences, Inc. The routine operations of Escherichia coli and Streptomyces lincomycin were carried out according to standard operating techniques. DNA sequencing was commissioned to Beijing Liuhe BGI Co., Ltd.

[0024] Table 1 The present invention relates to strains and plasmids

[0025]

[0026]

[0027] Table 2 The present invention relates to primers

[0028]

[0029] Example 1

[0030] SLCG_2790 gene related information:

[0031] Based on the gene annotation information of KEGG database (https: / / www.kegg.jp / ), the location information of SLCG_2790 and its surrounding genes in the genome of S. lincomyces and their potential gene functions are as follows Figure 1 shown.

[0032] The nucleotide sequence of the SLCG_2790 gene is shown in SEQ ID NO.1, with a total length of 765 bp; the encoded amino acid sequence is shown in SEQ ID NO.2, consisting of 254 amino acids.

[0033] ATGAGCACCGACGTCAGCAGTGCGGAGAACGAGGGTGGGGCGAGTGTCCGTACCGCGCGCGTGCCCAAGTACTACCGCCTGAAGAAGCACCTGCTCGACATGACGGAGACGCAGTCCCCGGGCACGCCGGTACCGCCCGAGCGCACGCTCGCCGCCGAGTTCGACACCTCGCGCACCACCGTGCGCCAGGCCCTTCAGGAGCTGGTGGTCGAGGGGCGGCTGGAGCGCATCCAGGGCAAGGGCACCTTCGTCGCCAAGCCGAAGGTCTCGCAGGCCCTCCAACTCACCTCGTACACCGAGGACATGCGCGCCCAGGGTCTCGAACCCACCTCGCAGCTGCTGGACATCGGCTACGTCACCGCCGACGACCGCCTCGCCGAGCTCCTCGACATCACGGCCGGCGGGCGGGTGCTGCGCATCGAACGCCTGCGCATGGCCAACGGCGAGCCCATGGCCATCGAGACCACCCACCTCTCCGCCAAGCGCTTCCCCGCCCTGCGCAGATCGCTGGTCAAGTACACGTCCCTCTACACGGCGTTGGCCGAGGTCTACGACGTCCACCTCGCCGAGGCCGAGGAGACCATCGAGACCTCCCTGGCCACCCCCCGCGAGGCCGGCCTCCTCGGCACCGACGTCGGCCTGCCGATGCTGATGCTCTCCCGCCACTCCCTGGACCGCGACGGCGGACCGGTGGAGTGGGTGCGCTCGGTGTACCGGGGAGACAGGTACAAGTTCGTGGCGCGCTTGAAAAGGCCCGTGGACTGA(SEQ ID NO.1)

[0034] MSTDVSSAENEGGASVRTARVPKYYRLKKHLLDMTETQSPGTPVPPERTLAAEFDTSRTTVRQALQELVVEGRLERIQGKGTFVAKPKVSQALQLTSYTEDMRAQGLEPTSQLLDIGYVTADDRLAELL DITAGGRVLRIERLRMANGEPMAIETTHLSAKRFPALRRSLVKYTSLYTALAEVYDVHLAEAEETIETSLATPREAGLLGTDVGLPMLSRHSLDRDGGPVEWVRSVYRGDRYKFVARLKRPVD(SEQ ID NO.2)

[0035] Example 2

[0036] Construction of SLCG_2790 knockout strain:

[0037] Design and select the appropriate sgRNA sequence of the SLCG_2790 gene on the sgRNA design website, and design primers based on the selected sgRNA. Using the pKCCas9do plasmid as the amplification template, 2790-P2 / P3 as primers were used to amplify the fragment containing the sgRNA and gRNA scaffold; then the amplified product was used as the amplification template, 2790-P1 / P3 as primers were used to add homologous sequences upstream of the fragment, and the DNA gel recovery kit was used for recovery, and the concentration was determined using Nanodrop. Using the L-427 genome as the amplification template, 2790-P4 / P5 and 2790-P6 / P7 were used as primers to amplify 1.0 kb of the upstream and downstream homologous arms of SLCG_2790, respectively, and recovered and quantified. At the same time, the pKCCas9do plasmid was double-digested with SpeI / HindIII and then recovered and quantified. According to the quantitative results, the digested plasmid, sgRNA, upstream homology arm, and downstream homology arm were mixed in a ratio of 1:10:10:10, and connected at 50°C using a seamless cloning kit. Then, all the ligation products were transformed into Escherichia coli DH5α, spread on LB plates containing 0.1% Apr and cultured for 18 hours to grow a single clone. Finally, a single clone was selected to expand the culture in liquid culture medium and extract the plasmid to obtain the plasmid pKCCas9do-Δ2790.

[0038] The obtained pKCCas9do-Δ2790 plasmid was transformed into Escherichia coli ET12567 / pUZ8002, and spread on LB plates containing 0..05% Apr, 0.05% Kana and 0.05% Cl, and cultured for 22 hours to grow a single clone. Finally, a single clone was selected to expand the culture in liquid culture medium and the plasmid was extracted to obtain the ET12567 / pUZ8002 strain carrying the plasmid pKCCas9do-Δ2790.

[0039] The obtained ET12567 / pUZ8002 carrying the plasmid pKCCas9do-Δ2790 was transferred into L-427 by mycelial conjugation. After the conjugate was cultured at 37°C for 24 hours, strains sensitive to Apr were screened. The mutant strain L-427Δ2790 with SLCG_2790 gene deletion was further verified by PCR. The identification primers used were 2790-P8 / P9. The schematic diagram of double exchange is shown in Figure 2 The results of genomic PCR identification are shown in Figure 3 .

[0040] Example 3

[0041] Detection of lincomycin production at the shake flask level in L-427 and L-427Δ2790:

[0042] The L-427 and L-427Δ2790 strains were fermented and the lincomycin production was detected at the shake flask level.

[0043] The results are as follows Figure 4 As shown: Compared with the starting strain L-427, the lincomycin A production of L-427Δ2790 was increased by 37.7%.

[0044] Example 4

[0045] Detection of lincomycin production at the 5 L fermentation tank level of L-427 and L-427Δ2790:

[0046] The L-427 and L-427Δ2790 strains were fermented in a 5 L fermenter, and their wet volume and lincomycin A production were detected.

[0047] The results are as follows Figure 5 , Figure 6As shown in the figure: after the loss of SLCG_2790, the overall bacterial concentration of the L-427Δ2790 strain was significantly better than that of L-427 during the fermentation period, and it was significantly improved in the later stage of fermentation. In the early stage of production, L-427Δ2790 was basically the same as L-427. After 96 hours of fermentation, L-427Δ2790 was always higher than L-427, and the maximum yield could reach 3640.6 mg / L, which was 39.6% higher than L-427 (2607.8 mg / L).

[0048] The above is a detailed introduction to one embodiment of the present invention. This article uses specific examples to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the technical solutions and core ideas of the present invention; ordinary technicians in this field should understand that they can still modify the technical solutions recorded in the aforementioned embodiments, or replace some of the technical features therein with equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for increasing the yield of lincomycin by modifying the gene of Streptomyces lincomycin SLCG_2790, characterized in that: The SLCG_2790 gene in Streptomyces lincomycin is deleted by genetic engineering to obtain a high-yield strain of lincomycin, and the obtained strain is used to ferment and produce lincomycin; wherein the nucleotide sequence of the SLCG_2790 gene is shown in SEQ ID NO.

1.

2. The method for increasing lincomycin production by modifying the gene of Streptomyces lincomycin SLCG_2790 according to claim 1, characterized in that: The amino acid sequence encoded by the SLCG_2790 gene is shown in SEQ ID NO.

2.

3. The method for increasing lincomycin production by modifying the gene of Streptomyces lincomycin SLCG_2790 according to claim 1, characterized in that: The SLCG_2790 gene expression product negatively regulates lincomycin biosynthesis.

4. An application of the method for increasing lincomycin production by modifying the gene of Streptomyces lincomycin SLCG_2790 as claimed in any one of claims 1 to 3, characterized in that: The SLCG_2790 gene was knocked out in the industrial strain of Streptomyces lincomycin to obtain a high-yield mutant for lincomycin A production.

5. The use of the method for increasing lincomycin production by modifying the gene of Streptomyces lincomycin SLCG_2790 according to claim 4, characterized in that: The industrial strain of Streptomyces lincomyces is selected as Streptomyces lincomyces L-427.

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