Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Engineering strain for directly producing gernebcin and use thereof

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
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of this method is that there are many impurities, the product yield is low, and the production cost is high.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Engineering strain for directly producing gernebcin and use thereof
  • Engineering strain for directly producing gernebcin and use thereof
  • Engineering strain for directly producing gernebcin and use thereof

Examples

Experimental program
Comparison scheme
Effect test

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.

[0050] Fermentation medium: soluble starch 20, raw soybean powder 20g, glucose 10g, NH 4 Cl5g, CaCO 3 (light) 5g, MgSO 4 4 g, FeSO 4 0.05g, ZnSO 4 0.03g, MnCl 2 0.3g, soybean oil 0.6mL / 40mL, 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...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

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...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12P19/00A61K31/702A61P31/04C12R1/465
Inventor 夏焕章隋鑫余永红侯曦凡
Owner SHENYANG PHARMA UNIVERSITY
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com