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Construction of biosynthesis gentamicin X2 engineering bacteria and application thereof

A technology of gentamicin and biosynthesis, applied in the direction of microorganism-based methods, microorganisms, biochemical equipment and methods, etc., can solve problems such as difficulties

Inactive Publication Date: 2014-05-28
FUZHOU UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The biosynthesis process of gentamicin contains many important amino-oligosaccharides, but they are only intermediates in the biosynthesis of amino-oligosaccharides. It is very difficult to obtain these important compounds, and breakthroughs are rare at home and abroad.

Method used

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  • Construction of biosynthesis gentamicin X2 engineering bacteria and application thereof
  • Construction of biosynthesis gentamicin X2 engineering bacteria and application thereof
  • Construction of biosynthesis gentamicin X2 engineering bacteria and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] 1. Construction of the shuttle plasmid pFU503

[0033] According to the gentamicin biosynthetic gene cluster sequence (GenBank Accession Number JQ975418) constructed by our laboratory, using Verctor NTI 11.5 software, respectively in genB3 To amplify the exchange arm upstream and downstream of the gene, design two pairs of primers, named P21 / P22, P23 / P24 respectively. p21(5'-GAATTC-CGTGTACGTCCCCTGAATCC-3', Eco RI), P22 (5'-CTCGAGGGGAGATCCTGACCAGTGAG-3', Xhol I), P23 (5'-CTCGAG TACAACAGCTCCGGCGAGAT-3', Xhol I), P24 (5'-TCTAGA TACGTCAACGTGCACGGGGT-3', Xba I). Chromosomal DNA of Micromonospora crimson was extracted, using the chromosomal DNA as a template, using primers P21 / P22 to amplify the upstream exchange arm JHB1 with a length of 2141bp. The PCR amplified product was recovered by DNA kit and cloned into pMD19-T to obtain a positive clone named pFU501; using primers P23 / P24, the downstream exchange arm JHB2 was amplified with a length of 2289bp. The PCR ampli...

Embodiment 2

[0040] Preparation of Metabolites of Micromonospora Crimson GKB3226

[0041] 1. Fermentation and Culture of Micromonospora Crimson GKB3226 Strain

[0042] 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.

[0043] 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.

[0044] Micromonospora magenta GKB3226 obtained in step 3 of Example 1, before fermentation, isolate a single colony with abundant sporulation by the dilution plate method, transfer it to a slant medium, cultivate it at 37°C for 10 days, and dig a block and inoculate it in the seed culture base (50mL / 250mL Erlenmeyer flask), cultured on a shaker at 37°C for 36 h (rotating at 250rpm). 10% of the inoculum was transferred to the fermentation medium (50mL / 250mL Erlenmeyer flask), and fermented at 37°C for 120 h (ro...

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Abstract

The invention belongs to the technical field of medicine, and relates to construction of biosynthesis gentamicin X2 gene engineering bacteria and application thereof. By the adoption of a microorganism molecular genetics technology, biosynthesis key genes of gentamicin in micromonospora purpurea are inactivated, gentamicin biosynthesis metabolic flux is cut off, and gentamicin X2 is directionally accumulated. By constructing recombinant plasmid pFU503 and leading pFU503 into micromonospora purpurea GK1101, the function of (i) genB3 ( / i) is inactivated through an in-frame knockout technology, double exchange bacterial strains are screened, fermentation products are detected, new components are identified, and gentamicin X2 producing bacteria are fixed. The constructed engineering bacteria have stable hereditary features, can accumulate a large number of gentamicin X2, and fill the foreign and domestic research gap. The constructed engineering bacteria production technology is simple, fermentation control is easy, the synthetic efficiency is high, extraction and purification are convenient, the product quality is stable, clean production state is nearly achieved, the bacteria have an important industrial application value, and great economic benefit can be produced.

Description

technical field [0001] The invention belongs to the field of antibiotic pharmacy, and relates to the construction and application of biosynthetic gentamicin X2 genetically engineered bacteria. It specifically involves the use of molecular genetics techniques to knock out Micromonospora rubrum M. purpurea Key genes of gentamicin biosynthesis in GK1101 genB3 , blocking the subsequent modification of gentamicin X2, accumulating gentamicin X2, which can be applied to drug development research. Background technique [0002] Microorganisms are an important source of medicines and the basis of microbial pharmaceuticals. Micromonospora can produce many important antibiotics and microbial enzymes, and its synthetic aminoglycoside antibiotics have important clinical application value. Since Waxman discovered streptomycin, aminoglycoside antibiotics have been used clinically It has been used for 70 years, and it is still an indispensable anti-infective drug in clinical practice. A...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N15/74C12P19/50C12R1/01
Inventor 洪文荣张熠林强
Owner FUZHOU UNIV
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