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Construction of geldanamycin gene engineering high yield strain

A technology of geldanamycin and high-yield strains, applied in the direction of microorganism-based methods, fungi, microorganisms, etc., can solve problems that have not been reported before, and achieve significant social and economic benefits

Inactive Publication Date: 2007-10-17
MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The method for constructing geldanamycin high-yield bacterial strains using gene blocking technology in the present invention has not been reported so far

Method used

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  • Construction of geldanamycin gene engineering high yield strain
  • Construction of geldanamycin gene engineering high yield strain
  • Construction of geldanamycin gene engineering high yield strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Construction of naphthoquinone type AHBA gene blocking recombinant vector

[0024] Primers were arbitrarily designed according to the shn SOP gene sequence, and the genomic DNA of Streptomyces hygroscopicus 17997 was extracted, which was used as a template for conventional PCR. In this experiment, the following primers and designed restriction sites were used, but not limited to the sequences of the primers.

[0025] Upstream primer: P1 5'-CCG GAATTC ACACTCGCACGTCCAGCC-3' has an EcoRI restriction site;

[0026] Downstream primer: P2 5'-GCG GGTACC GCAGCCAGATGCAGTCGG-3' has a KpnI restriction site; amplifies the 1257bp fragment S1.

[0027] Upstream primer: P3 5'-AAAA CTGCAG CTGTGGCTCAGACTGCTGC-3' has a PstI restriction site;

[0028] Downstream primer: P4 5'-CTAG TCTAGA TGATGTCGGAAAGTAGCG-3' has an XbaI restriction site; amplifies the 1265bp fragment S2.

[0029] The PCR primers designed in the present invention are shown in Figure 1 at the corresponding parts...

Embodiment 2

[0031] Obtaining and identification of gene blocking strain

[0032] The screening of gene-blocking strains was carried out according to the literature [He Weiqing et al., Chinese Journal of Antibiotics, 2006, 31(3): 168-171]. Introduce the constructed gene blocking vector pGEX-shn into Streptomyces hygroscopicus 17997 through Escherichia coli capable of conjugative transfer with Streptomyces hygroscopicus, such as Escherichia coli ET12567 / pUZ8002 [John InnesCentre, Norwich Research Park, Colney Norwich R4 7UH, UK] , and passed 3 to 4 generations in MY medium, and then isolated the monoclonal, according to the Tsr and Am resistance phenotype, screened to obtain the blocking mutant strain ΔSOP that was Am resistant and Tsr sensitive through double exchange of homologous genes . The ΔSOP blocking mutant was verified by PCR at the level of gene integration. Using the genomes of the original strain and the ΔSOP variant strain as templates, select primers P5 and P6 (see Figure 1...

Embodiment 3

[0036] The improvement of geldanamycin fermentation yield in ΔSOP variants

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Abstract

The present invention relates to geldanamycin high yield engineered bacteria strain construction, It is specificly described as follows: two groups of AHBA synthase gene cluster AHBA-shn gene discovered in water-absorbing streptomyces are destroyed by gene blocking technology due to likely participation in naphthoquinone type ansamycin compound biosynthesis. The present invention increases benzenquinone type GDM precursor (B-AHBA) supply and metabolic flow to promote GDM ferment yield by blocking or reduceing content of naphthoquinone type ansamycin compound precursor (N-AHBA). It proves that block mutant strain promote near 186% in yield to original strain.

Description

Technical field: [0001] The invention relates to the application of genetic engineering technology in the construction of antibiotic high-yield bacterial strains. Background technique: [0002] Geldanamycin (geldanamycin, GDM) producing bacteria - Streptomyces hygrocopicus 17997 (Streptomyces hygrocopicus 17997), is the Chinese Academy of Medical Sciences Institute of Pharmaceutical Biotechnology isolated from Chinese soil [Tao Peizhen and other Chinese Journal of Antibiotics, 1997, 22( 5): 368]. Geldanamycin has antitumor and broad-spectrum antiviral effects. Recent studies have shown that GDM can specifically inhibit the function of heat shock protein 90 (Hsp90). Hsp90 is a chaperone molecule for many signaling proteins. GDM can indirectly affect the function of intracellular signaling proteins by inhibiting Hsp90. Therefore, GDM is expected to become a potential anti-tumor and anti-viral drug. The novel mechanism of action of GDM determines the potential scientific an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/15C12P17/10C12R1/55
Inventor 王以光赫卫清高群杰
Owner MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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