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A kind of engineering bacteria producing g418 single component and its application

An engineering bacteria, single-component technology, applied in bacteria, recombinant DNA technology, microorganism-based methods, etc., can solve the problems of high price, difficult preparation of G418, complicated process, etc., achieve convenient extraction and purification, fill research gaps, fermentation Control simple effects

Inactive Publication Date: 2016-03-30
FUZHOU UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The preparation of G418 is extremely difficult, requires chromatographic separation, and the process is complicated. Therefore, the price is expensive, and all G-418 needed in China depends on imports

Method used

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  • A kind of engineering bacteria producing g418 single component and its application
  • A kind of engineering bacteria producing g418 single component and its application
  • A kind of engineering bacteria producing g418 single component and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] 1. Construction of homologous recombination plasmid pQB303

[0029] According to the gentamicin biosynthesis gene cluster (GenBankaccessionNo.: JQ975418, 44181bp) of Micromonas magenta published by Hong Wenrong et al. in 2012 and the gentamicin biosynthesis gene cluster of Micromonas aculeatus published by Piepersberg et al. GenBankAccessionNumberAJ628149), with gene gen Q Upstream and downstream sequences, design an in-frame knockout scheme ( figure 2 ). The two pairs of amplification exchange arm primers are PQ1 / PQ2 and PQ3 / PQ4 respectively. P1(5'-GAATTCAGGAGGTGCTCACCGACG-3', Eco RI), P2 (5'-AAGCTTAGAACCGGGTGTCCCTCG-3', Hin dIII); P3 (5'-AAGCTTTTCCGTTCGAAGGCGACC-3', Hin dIII), P4 (5'-TCTAGAAACGGCTCGGTGAACTCGTG-3', Xba I).

[0030]Using the genome of Micromonospora rubrum G1008 as a template, the PCR reaction system and reaction conditions are: 95°C for 5 minutes, 94°C for 1 minute, 67.5°C for 1 minute, 72°C for 1 minute, 72°C for 10 minutes, and 4°C for st...

Embodiment 2

[0038] Embodiment 2: Preparation of engineering bacteria GQ175 metabolites

[0039] 1. Fermentation of Engineering Bacteria GQ175 Strain

[0040] Seed medium: glucose 0.1%, corn starch 1.0%, corn flour 1.5%, peptone 0.2%, soybean meal 1.0%, KNO 3 0.05%, CaCO 3 0.5%, pH7.0.

[0041] Fermentation medium: corn starch 6.0%, corn flour 1.0%, peptone 0.4%, soybean cake powder 2.0%, KNO 3 0.01%, (NH 4 ) 2 SO 4 0.1%, CaCO 3 0.5%, amylase 0.025%, pH7.5.

example 1

[0042] Fermentation of Micromonospora magenta GQ175 obtained in Example 1 step 3. Before fermentation, isolate the single colony with rich sporulation by dilution plate method and transfer it to the slant medium, culture at 37°C for 10 days, excavate the block and inoculate it into the seed medium (50mL / 250mL Erlenmeyer flask), and culture on a shaker at 37°C 36h (rotating speed: 250rpm), when submerged culture enters the logarithmic growth phase, transfer 10% of the inoculum to the fermentation medium (capacity: 50mL / 250mL Erlenmeyer flask), and ferment on a shaking table at 37°C for 120h (rotating speed: 250rpm).

[0043] 20,000 liters fermenter production, stirring speed 180 rpm, ventilation volume 1: 0.5~1.0(M 3 / M 3 min), culture medium, culture temperature, inoculum ratio, fermentation time, etc., similar to shake flask fermentation.

[0044] 2. Extraction and purification of metabolites

[0045] A. Rough extraction

[0046] After the fermentation broth is diluted an...

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Abstract

The invention belongs to the pharmaceutical field of antibiotics and relates to an engineering strain for producing a G418 single component and an application thereof. The engineering strain is micromonospora purpurea GQ175, the strain is registered and collected by China General Microbiological Culture Collection Center (CGMCC) on December 6, 2013, and the collection number is CGMCC No. 8543. A gentamicin complex is not synthesized any more by damaging biosynthesis key genes of gentamicin in the micromonospora purpurea, blocking a biosynthesis metabolic flux and promoting the accumulation of a metabolite intermediate G418. According to the engineering strain provided by the invention, a gentamicin biosynthesis gene (genQ) in the micromonospora purpurea is killed through a knocking-out method in a gene frame, a shuttle vector pDB303 is constructed, the pDB303 is transformed to micromonospora purpurea G1008, a double-exchange strain is screened, fermentation production is performed, and detection of a metabolite and identification of new components are further performed. The engineering strain provided by the invention can produce the single component G418 so as to fill in the blank that the G418 can not be produced at home, and the engineering strain has the advantages of simple production process, low cost, high yield, convenience in control and stable quality.

Description

technical field [0001] The invention belongs to the field of antibiotic pharmacy, relates to an engineering bacterium producing G418 single component and its application, and utilizes molecular biology operation technology to clarify gen Q (GenBankaccessionNo.: AJ628149, gen Q ; AY524043.1, gntX; AJ575343.3, gacJ) gene function, and disrupt the key gene of amino-oligosaccharide biosynthesis in Micromonospora rubrum G1008 gen Q , so as to obtain a high-yield G418 engineering bacteria, which can be applied to the production of amino oligosaccharides and produce G418. Background technique [0002] Aminoglycoside antibiotics are antibiotic drugs widely used clinically, including streptomycin, neomycin, kanamycin and gentamycin, etc. These compounds rely on their own chemical structure characteristics and bind to the 30S ribosomal subunit. Different regions of the base interfere with bacterial protein synthesis, thereby killing or inhibiting bacterial growth. Aminoglycoside an...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N15/74C12P19/50C12R1/31
Inventor 洪文荣阙新桥胡育龙
Owner FUZHOU UNIV
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