Genetically engineered bacteria for producing lincomycin as well as construction method and application of genetically engineered bacteria

A genetically engineered bacterium and a technology for producing lincomycin, applied in the field of genetic engineering

Active Publication Date: 2020-05-08
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no case of improving l...

Method used

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  • Genetically engineered bacteria for producing lincomycin as well as construction method and application of genetically engineered bacteria
  • Genetically engineered bacteria for producing lincomycin as well as construction method and application of genetically engineered bacteria
  • Genetically engineered bacteria for producing lincomycin as well as construction method and application of genetically engineered bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0126] Example 1 Construction of slinc566, slinc4481, slinc6027, slinc6156, slinc348, slinc4742 knockout vectors

[0127] The gene knockout scheme in this example is based on CRISPR / Cas9 technology.

[0128] The PCR reaction conditions of this embodiment are: 98°C, 10 minutes; (98°C, 40 seconds; 64°C, 30 seconds; 72°C, 80 seconds) × 30 cycles; 72°C, 10 minutes; 25°C, 2 minutes .

[0129] 1. Construction of slinc566 knockout vector

[0130] Step 1: Using u566-F / R and d566-F / R as primers and Streptomyces lincolnensis NRRL 2936 genome as a template, PCR amplifies the upstream and downstream homology arms of slinc566. After detection by 1% agarose gel electrophoresis, the target band at 1.1 kb was cut and purified.

[0131] Step 2: Use the upstream and downstream homology arm fragments obtained in the previous PCR reaction as templates, and use sg566 / u566-R as primers to amplify by PCR to add the sgRNA sequence that specifically recognizes the sequence within the slinc566 gene....

Embodiment 2

[0143] Example 2 Gene knockout of slinc566, slinc4481, slinc6027, slinc6156, slinc348, and slinc4742.

[0144] The PCR reaction conditions of this embodiment are: 98°C, 10 minutes; (98°C, 40 seconds; 64°C, 30 seconds; 72°C, 80 seconds) × 30 cycles; 72°C, 10 minutes; 25°C, 2 minutes .

[0145] 1. The principle of slinc566 gene knockout of the present invention is as follows: Figure 7 shown.

[0146] Step 1, transforming the pKCcas9d566 plasmid into Escherichia coli S17-1. Cultured in LB medium to OD 600 =0.4 pKCcas9d566 / S17-1 and Streptomyces lincolnensis NRRL2936 cultured in YEME medium until the hyphae were uniform and dense, collected by centrifugation and washed, resuspended in 2×YT medium, and spread on ISP4 medium together, at 28°C Cultured for conjugative transfer. After 18 hours of plate coating, 1 mL of sterile aqueous solution containing apramycin (Apramycin, 20 μg / mL) and nalidixic acid (Nalidixic acid, 25 μg / mL) was covered for screening. After continuing to ...

Embodiment 3

[0163] Example 3 Construction of genetically engineered bacteria Δ4481Δ6156

[0164] The PCR reaction conditions of this embodiment are the same as that of embodiment 2.

[0165] Step 1: The pKCcas9d6156 plasmid was transformed into Escherichia coli S17-1. Cultured in LB medium to OD 600 =0.4 pKCcas9d6156 / S17-1 and the genetically engineered strain Δ4481 that was cultured in YEME medium until the hyphae were uniform and dense, collected by centrifugation and washed, resuspended in 2×YT medium, and spread on ISP4 medium together, at 28°C Cultured for conjugative transfer. After 18 hours of plate coating, 1 mL of sterile aqueous solution containing apramycin (Apramycin, 20 μg / mL) and nalidixic acid (Nalidixic acid, 25 μg / mL) was covered for screening. After continuing to culture at 28°C for 4-7 days, colonies of transformants can grow. Pick 10 transformant mycelia and transfer them to MS medium containing 20 μg / μL apramycin, continue culturing at 28°C for 3-5 days, then inoc...

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Abstract

The invention discloses genetically engineered bacteria for producing lincomycin. The genetically engineered bacteria are constructed by knocking out a gene for encoding protein containing a tetratricopeptide repeat (TPR) structural domain of streptomyces lincolnensis, knocking out a gene for encoding an RNA enzyme and performing overexpression on a gene for encoding Xre family protein of the streptomyces lincolnensis. The invention also discloses a construction method of the genetically engineered bacteria for producing the lincomycin. The method comprises the steps of knocking out the gene for encoding the protein containing the TPR structural domain of the streptomyces lincolnensis by using the streptomyces lincolnensis as a starting strain, knocking out the gene for encoding the RNA enzyme and performing overexpression on the gene for encoding the Xre family protein of the streptomyces lincolnensis to construct the genetically engineered bacteria for producing the lincomycin. The construction method of the genetically engineered bacteria for producing the lincomycin provided by the invention is easy and convenient to operate, and the obtained genetically engineered bacteria canincrease the yield of the lincomycin and have good application prospects.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a genetically engineered bacterium producing lincomycin and its construction method and application. Background technique [0002] Lincomycin is a lincosamide antibiotic produced by Streptomyces lincolnensis with wide clinical value. Lincomycin and its derivatives (such as the chlorinated derivative clindamycin) have inhibitory effects on anaerobic Gram-positive bacteria and protozoa. Lincomycin inhibits the transfer of polypeptide chains during protein translation by hydrophobically binding to the central loop of 23s rRNA in the 50s subunit of ribosomes, thereby exerting its antibacterial function. Lincomycin does not have cross-resistance to most pharmaceutical antibiotics, and is widely used clinically in the treatment of respiratory tract, urinary system, skin and soft tissue infections, chronic osteomyelitis, meningitis, otitis media and eye diseases, etc., es...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/76C12N15/90C12N15/55C12N15/31C12R1/565
CPCC07K14/36C12N9/22C12N15/76C12N15/902
Inventor 吴海珍叶江张惠展侯兵兵王瑞达
Owner EAST CHINA UNIV OF SCI & TECH
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