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Sisomicin-producing micromonospora engineering bacteria and its construction and application

A technology of Micromonospora crimsonosa and sisomicin, which is applied to engineering bacteria producing sisomicin Micromonospora crimson and its construction and application, can solve problems such as elucidating the regulation mechanism of biosynthesis, etc. Achieve the effect of reducing the extraction production process, reducing the cost of fermentation production, and high productivity

Active Publication Date: 2019-03-15
福州市鼓楼区荣德生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] But the main difficulty is that at present, the functions of all the genes in the biosynthesis gene clusters of gentamicin and sisomicin have not been fully elucidated at home and abroad, let alone their biosynthesis regulation mechanism

Method used

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  • Sisomicin-producing micromonospora engineering bacteria and its construction and application
  • Sisomicin-producing micromonospora engineering bacteria and its construction and application
  • Sisomicin-producing micromonospora engineering bacteria and its construction and application

Examples

Experimental program
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Effect test

Embodiment 1

[0026] Gene genB4 Explanation of function:

[0027] Using the gentamicin biosynthesis gene cluster registered in Genebank (accession number: JQ975418) as a template, two pairs of primers were designed using the bioinformatics software Vector NTI11.5, and amplified genB4 The upstream and downstream sequences of the gene are used as homologous exchange arms: primers P1 / P2 are used to amplify the upstream exchange arm B41, and primers P3 / P4 are used to amplify the downstream exchange arm B42. Design primers P5 / P6 for genB4 Detection of gene blockade mutants. The primers and the length of the amplified fragment are as follows: figure 2 See Table 1 for primer sequences and their restriction enzyme sites.

[0028] Table 1 Genes genB4 Functional research related primers

[0029]

[0030] 1 Construction of recombinant plasmid pGB403

[0031] Genomic DNA of Micromonospora magenta G1008 was extracted as a template. PCR amplification genB4 Swap arms B41 and B42 upstream and...

Embodiment 2

[0043] Breeding of Strains with High Yield of Gentamicin C1a

[0044] Using gentamicin-producing strain G1008 as the starting strain, through ultraviolet mutagenesis, isolation and purification of single colony, fermentation, extraction, determination of metabolite structure and other tests, the mutant strain with gradually increased gentamicin C1a component was determined. After 48 generations and more than three years of directional breeding, the main gentamicin C1a sub-starter strain Micromonospora crimson Gb was obtained, which was used for the next step of gene recombination to construct a single-component sisomicin-producing engineering bacterium.

Embodiment 3

[0046] Construction of GbKB4 Engineering Bacteria

[0047] According to the method similar to Example 1, the plasmid pFD306 (the patent of this research series: 201110331534.9) was introduced into the donor bacteria to obtain the donor bacteria E. coli ET12567 / (pFD306 / pUZ8002). Conjugate the donor bacteria with the spores of Micromonospora crimsonum Gb. After 7 days, the zygotes grow on the MS plate, pick a well-growing strain and name it GbK. Transfer it to Micromonospora seed culture medium, isolate a single colony after 4 generations of relaxation culture, and copy it to a plate containing 50 µ / mL apramycin and no antibiotics, and screen to obtain the engineering bacteria GbK (△ Gen K ), extract its genomic DNA, use primers P5 / P6 for PCR verification, electrophoresis detection and DNA sequencing, and confirm that it is the target engineering bacteria.

[0048] Knock out △ as above Gen K The method of introducing the plasmid pGB403 into the donor bacteria E. coli E...

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Abstract

The invention provides a sisomicin-producing micromonospora purpurea engineering bacterium, and construction and application thereof, and belongs to the field of microbe pharmacy, and clarifies genB4 gene function by means of gentamycin-producing micromonospora purpurea G1008. The gentamycin-producing micromonospora purpurea G1008 is selectively bred, and an original strain Gb taking gentamycin C1a metabolism approach as the main approach is obtained. By constructing shuttle plasmids pFD306 and pGB403 knocking out genK and genB4 and successively introducing the shuttle plasmids pFD306 and pGB403 into micromonospora purpurea Gb and screening zygotes, the sisomicin-producing micromonospora purpurea GB4408 is obtained, and is characterized by possessing the unique heredity characteristic of highly producing sisomicin biosynthesis potential. Construction of the engineering bacterium is not reported at home and abroad, and application to prepare sisomicin antibiotic medicaments is satisfied.

Description

technical field [0001] The invention belongs to the field of microbial pharmacy, and relates to micromonospora engineering bacteria producing sisomicin crimson red and its construction and application. The invention uses molecular biology operation technology to clarify genB4 The function of the gene, by constructing the recombinant plasmid pGB403, and transforming the shuttle plasmid pGB403 E. coli ET12567 (pUZ8002) obtained conjugative transfer donor bacteria E. coli ET12567 (pGB403 / pUZ8002), screened the zygote, and obtained the sisomicin-producing engineering strain Micromonospora crimson GB4408, which has unique genetic characteristics of high-yield sisomicin biosynthetic potential, which can be applied to sisomicin manufacture. Background technique [0002] Amino oligosaccharides are important anti-infective drugs with important clinical application value and unique antibacterial activity. After more than half a century of long-term clinical application and test...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/74C12P19/48C12R1/31
Inventor 洪文荣
Owner 福州市鼓楼区荣德生物科技有限公司
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