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Engineering bacterium capable of producing anthracene ring antibiotics and application of the same

A technology of engineering bacteria and anthracyclines, applied in the field of genetic engineering

Active Publication Date: 2007-08-15
ZHEJIANG HISUN PHARMA CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The technical problem to be solved in the present invention is to construct a gene blocking mutant of a daunorubicin producing bacterium Streptomyces coeruleorubidus that cannot directly produce daunorubicin, and to introduce Some specific genes can rebuild metabolic pathways to produce anthracycline antibiotics, such as epidaunorubicin and other anthracycline antibiotics to produce engineering bacteria

Method used

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  • Engineering bacterium capable of producing anthracene ring antibiotics and application of the same
  • Engineering bacterium capable of producing anthracene ring antibiotics and application of the same
  • Engineering bacterium capable of producing anthracene ring antibiotics and application of the same

Examples

Experimental program
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Effect test

Embodiment 1

[0047] Example 1 Cloning and sequencing of dnmU and dnmV linked genes of Streptomyces coelicolor SIPI-1482

[0048] dnmU in Streptomyces coelicolor is linked to and upstream of the dnmV gene. In order to achieve directional blocking of the dnmV gene, primers were first designed according to the published corresponding sequence of S. peucetius (Accession Number AF006633), respectively dnmUV1: 5'-AA CTG CAG GAGCGAAGTGGCGTTG-3', containing PstI site; dnmUV2: 5'-GG GAATTC TCGTCGGAAGCCTGTG-3' with EcoRI site. The PCR reaction conditions were as follows: denaturation at 97°C for 5 minutes, adding Taq enzyme, 1 minute at 95°C, 1 minute at 61°C, 3 minutes at 72°C, and extension at 72°C for 10 minutes after 30 cycles. The dnmU and dnmV linkage fragments were amplified using the total genome of Streptomyces coelicolor SIPI-1482 as a template, and the full length of the PCR product was 1664bp (as shown in Figure 1). The dnmU and dnmV linked fragment PCR products were ligated to pUC...

Embodiment 2

[0049] Example 2 Construction, Transformation and Blocker Screening of Double Crossover Plasmids

[0050] The plasmid pYG803 (4299bp) prepared in Example 1 was digested with KpnI and NotI, and a 4080bp fragment was recovered. Combine this fragment with the apramycin resistance gene fragment (published in GenBank, about 1.3kb, Accession number: AY072040, AY216678, AJ438947, AJ566337) that was also digested with KpnI and NotI and use T4 ligase at 16°C After 4 hours of ligation, E.coliDH5a was transformed. Apramycin and ampicillin were used as selection markers to jointly screen the target clones, pick transformants, extract plasmids for enzyme digestion verification, and successfully construct the double-crossover plasmid pYG817 (5466bp) (as shown in Figure 3).

[0051] Alkaline denaturation and single-strandization of the obtained plasmid, that is, 110μl plasmid dilution solution system, the concentration is about 50ug / ml, 1:1 adding 0.4M NaOH to make the final concentration 0...

Embodiment 3

[0052] Example 3 Screening of double-exchange engineering bacteria

[0053] The spores of the regenerated colonies were inoculated into 20 ml of YMB medium, cultured and collected, and their chromosomes were extracted. According to the apramycin resistance gene sequence (Accession number: AY072040, AY216678, AJ438947, AJ566337) published in GenBank, its homology was compared, and primers were designed for regions with higher homology: apr1: 5'-GATGCAGGAAGATCAACG- 3'; apr2: 5'-AGGTCTGGACGACGAGC-3'. The genomic DNA of the regenerating bacteria and the genomic DNA of Streptomyces coelicolor SIPI-1482 (control strain) were respectively used as templates for PCR amplification. As a result, a fragment of about 500 bp inside the apramycin resistance gene could be amplified from the genome of the regenerating bacteria. However, no fragments could be obtained from the genome of the control strain (as shown in FIG. 4 ).

[0054] Also use the primers dnmV1 on both sides of the dnmV gen...

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Abstract

The invention discloses an engineering bacterium through anthracene nucleus antibiotic and appliance, which is characterized by the following: inserting or replacing restriction enzymes site of dnmV gene nucleic acid order of sky blue rufus streptomycete with antibiotic against property gene and surface cerubidin synthetic gene; blocking-up engineering bacterium of dnmV gene; or transforming expressing carrier of anthracene nucleus antibiotic synthetic gene to lead streptomycin (S.lividans)TK24; extracting expressing plasmid from S.lividans TK24; leading-in sky blue rufus streptomycete gene only with antibiotic against property gene to block dnmV gene; blocking-up abrupt change bacterium. This invention can produce cerubidin and / or analogue, such as anthracene nucleus antibiotic.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to an engineering bacterium for producing anthracycline antibiotics and its application. Background technique [0002] Daunorubicin and doxorubicin and epirubicin synthesized from it are currently clinically very important anti-tumor anthracycline antibiotics. Epirubicin is less toxic to the heart and bone marrow than doxorubicin and has comparable or greater antineoplastic effects. Epi-daunorubicin (epi-daunorubicin) is an important precursor for industrial production of epirubicin. The difference between it and daunorubicin lies in the configuration of the hydroxyl group at the C4 position of the deoxysugar in the molecule. The chemical synthesis from daunorubicin to epidaunorubicin needs to go through a multi-step reaction process with harsh conditions and will cause environmental pollution. If the microbial fermentation method is used for direct production, the c...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/31C12P17/14C12R1/465
Inventor 朱宝泉朱春宝尚珂宫倩胡又佳
Owner ZHEJIANG HISUN PHARMA CO LTD
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