Engineering strain for directly producing gernebcin and use thereof
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A technology of tobramycin and engineering bacteria, applied in the directions of microorganism-based methods, medical preparations containing active ingredients, bacteria, etc., to achieve the effects of reducing production costs, facilitating quality control, and simplifying production processes
Inactive Publication Date: 2013-05-15
SHENYANG PHARMA UNIVERSITY
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The disadvantage of this method is that there are many impurities, the product yield is low, and the production cost is high.
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Embodiment 1
[0028] The present invention mainly comprises following major steps:
[0029] 1. Construction of tacA gene blocking plasmid
[0030] According to the sequence of the tobramycin biosynthetic gene cluster (GenBank Accession Number AJ579650) published by Madan Kumar Kharel et al., two pairs of primers were designed on the upstream and downstream of the tacA gene, and two fragments were obtained by PCR amplification using the total DNA of Streptomyces fugus as a template PCR1-2 and PCR3-4. The two-fragment ligation product was named ΔtacA, which deleted 111 bases at the 5' end, 260 bases inside and 88 bases at the 3' end of the tacA gene. ΔtacA was ligated into vector pIJ2925 to obtain pSPU301. The erythromycin resistance gene ermE was then connected to the KpnI site in pSPU301 (pXZ1 was digested with KpnI to recover a fragment of about 1.6 Kb in size) to obtain the recombinant plasmid pSPU302, and finally the ΔtacB+ermE was connected to BamHI in the vector pHZ132 by digesting w...
Embodiment 2
[0046] Embodiment 2: utilize tobramycin genetically engineered bacteria to produce tobramycin
[0047] The tobramycin genetically engineered bacterium provided by the invention can be directly used in production, and the tobramycin is extracted after the strain is fermented as an antibacterial drug.
[0048] 1. Shake flask fermentation of tobramycin genetically engineered bacteria
[0049] Seed medium: raw soybean powder 10g, glucose 10g, peptone 3g, yeast powder 1g, corn flour 5g, CaCO 3 (Light weight) 1g, add tap water to 1L.
[0051] The genetically engineered bacterium Streptomyces fugus H6 (pSPU303-3) obtained in step 3 of Example 1 was subjected to shake-flask fermentation. Before the fermentation, the single colony with rich sporulation was isolat...
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Abstract
The invention pertains to the field of medical technology and relates to engineering bacteria directly producing tobramycin and the application thereof, which mainly damages carboxamide transferase gene in bacteria produced by the tobramycin, therefore the strain does not produce carboxamide tobramycin any more but tobramycin. The invention, by the method of deleting inactivation within the frame, breaks tacA gene in Streptomyces tenebrarius, comprising the composition of the tacA gene breaking plasmid, the breaking of the transformation of plasmid pSPU303 into Streptomyces tenebrarius H6, the screening of double exchange strains, the detection of fermentation products and the identification of new compositions. The method of deleting inactivation within the frame respectively provides two segments with the upper reach molecular weight and the lower reach molecular weight of the tacA gene not less than 500bp for the PCR, both segments are connected to pIJ2925 simultaneously, one end of the segment is connected to resistance gene of antibiotics that is expressible in the Streptomyces tenebrarius such as erythromycin resistance gene ermE, and three gene segments are connected to shuttle vector pHZ132 by using Bg1II Enzyme cutting. The engineering bacteria directly producing tobramycin and the application thereof can simplify the production technology and reduce the production cost, thus facilitating the quality control of the products.
Description
technical field [0001] The invention belongs to the field of antibiotic pharmacy, and relates to an engineering bacterium directly producing tobramycin and its application. Specifically, the invention uses molecular biology operation technology to block the biosynthetic gene tacA of Streptomyces tenebrarius , to obtain a genetically engineered strain directly producing tobramycin, which can be used in antibiotic pharmacy. [0002] Background technique [0003] Streptomyces tenebrarius was isolated from the soil in 1967 by researchers from the Eli Lilly Company of the United States. This strain can produce a group of antibiotics, of which 2, 4, and 5' are the main components. Component 2 is Apramycin (Apramycin, Am), Component 4 is 6”-O-Carbamoylkanamycin B (6”-O-Carbamoylkanamycin B, CKB), and Component 5’ is 6” -O-Carbamoyl tobramycin (6"-O-Carbamoyl tobramycin, CTB). Components 4 and 5' form kanamycin B and tobramycin respectively after high temperature hydrolysis. [0...
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