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Anthracene nucleus antibiotic producing strain dnrX gene fragment, gene breaking engineering bacterium prepared from the same and application thereof

A technology of gene fragments and antibiotics, applied in the field of genetic engineering, can solve problems such as low conversion rate, environmental pollution, complex process, etc., and achieve the effect of increasing production

Inactive Publication Date: 2012-04-25
SHANGHAI INST OF PHARMA IND CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The process is complicated, the conversion rate is low, and it causes environmental pollution

Method used

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  • Anthracene nucleus antibiotic producing strain dnrX gene fragment, gene breaking engineering bacterium prepared from the same and application thereof
  • Anthracene nucleus antibiotic producing strain dnrX gene fragment, gene breaking engineering bacterium prepared from the same and application thereof
  • Anthracene nucleus antibiotic producing strain dnrX gene fragment, gene breaking engineering bacterium prepared from the same and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1 PCR amplification dnrX' gene

[0033] Since only the dnrX gene sequence (accession number AF048833) derived from the daunorubicin-producing strain Streptomyces persei ATCC29050 has been published in GenBank, and there is no relevant report on the gene sequence outside the final codon. Given that the purpose of amplifying the gene is to block mutations, an incomplete gene can be amplified. Based on this, primers dnrX1 and dnrX2 were designed.

[0034] dnrX1 sequence: 5'-GGCCGCCAGCCGCTGTCCGA-3' (SEQ ID No.5)

[0035] dnrX2 sequence: 5'-GTGGTTCCAGGCGAAGAGCAGCGCATAGTC-3' (SEQ ID No.6)

[0036] PCR amplification was performed using the SIPI-1482 genome of S. coeruleorubidus as a template, and the reaction system was shown in Table 1:

[0037] Table 1 Composition of PCR reaction system

[0038]

[0039] The reaction conditions were: pre-denaturation at 97°C for 5 min, denaturation at 95°C for 0.5 min, annealing at 60°C for 1 min, extension at 72°C for 1 min,...

Embodiment 2

[0042] Preparation and sequencing of the gene fragment dnrX" of embodiment 2dnrX'

[0043] The plasmid pYG957 was digested with Nco I, and the large fragments were recovered for self-ligation, transformed into DH5α competent cells, and the intermediate plasmid pYG962 (such as Figure 4 shown). The intermediate plasmid pYG962 was verified by double digestion with Kpn I and HindIII, and the result of double digestion with Kpn I and HindIII of pYG957 was used as a control. Such as Figure 5 As shown, two bands of 1203bp and 2651bp were obtained by double enzyme digestion of pYG962, and two bands of 1080bp and 2651bp were obtained by double enzyme digestion of pYG957. correct.

[0044] Then, dnrX" was obtained by digestion with KpnI+HindIII from plasmid pYG962. The measured sequence is shown in SEQ ID No.3 in the sequence listing, and the encoded amino acid sequence is shown in SEQ ID No.4.

Embodiment 3

[0045] Example 3 Construction and Verification of Destroying dnrX Using Single Exchange to Block Mutation Plasmid pYG963

[0046] The dnrX" gene fragment obtained from plasmid pYG962 digested with KpnI+HindIII and the apramycin resistance gene apr (GenBank accession number: AJ566337, commercially synthesized by Yingjun Company) were connected to the vector pBluescript2KSM and constructed into recombinant plasmid pYG963 (Such as Figure 6 shown), transform DH5α competent cells, and select transformants by apramycin and ampicillin resistance. Extract the plasmid for PCR verification and enzyme digestion verification, such as Figure 7 shown. Using dnrX1 and dnrX2 as primers for PCR, using the plasmid pYG957 as a template can amplify the partial gene dnrX' (1080bp) of the dnrX gene, but not using pYG963, such as Figure 7 (A), consistent with the expected results. Use the common enzyme cutting site Sal I on the original vector and the insert to carry out pYG963 enzyme digesti...

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Abstract

The present invention discloses a nucleotide sequence which roots in an anthracycline and produces the partial gene dnrX1 and the gene fragment dnrX 2 of the dnrX 1 of streptomyces coeruleorubidus (Streptomyces coeruleorubidus), and an express vector containing the sequences. The present invention also discloses a gene disruption engineering strain which disrupts the treptomyces coeruleorubidus of the dnrX gene by adopting the express vectors with an exchange and homologous recombination method. When the gene disruption engineering strain is used to produce the anthracycline, the acidizing treatment is not required, adriacin which is not produced by a wild fungus can be produced, and the output of the adriacin and the daunorubicin can be greatly increased.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a dnrX gene segment of an anthracycline antibiotic-producing bacterium, a gene-blocking engineering bacterium prepared from the gene segment, and an application thereof. Background technique [0002] Anthracycline antibiotics, such as daunorubicin (DNR) and doxorubicin (DXR), are clinically used as first-line antineoplastic drugs, and doxorubicin is a derivative product of the C-14 hydroxylation of daunorubicin. Erythromycin has a wider anti-tumor spectrum and lower toxicity and side effects. At present, doxorubicin is produced industrially by chemical semi-synthesis, and doxorubicin is obtained from daunorubicin produced by microorganisms through a seven-step reaction. The process is complicated, the conversion rate is low, and it causes environmental pollution. [0003] Foreign studies on the biosynthetic pathway of daunorubicin-producing bacteria have shown that in the bio...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/31C07K14/37C12N1/15C12P19/56C12R1/465
Inventor 朱春宝宫倩胡又佳朱宝泉
Owner SHANGHAI INST OF PHARMA IND CO LTD
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