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Streptomyces diastatochromogenes engineering strain for producing toyocamycin, as well as construction method and application thereof

A technology for producing Streptomyces chromogenes and toyogamycin, which is applied in the field of genetic engineering and can solve the problems of complex protoplast transformation operations, low transformation efficiency, and uninspected effects.

Inactive Publication Date: 2013-08-14
CHINA JILIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the protoplast transformation operation is complex and requires the preparation of protoplasts. The protoplast transformation also has a host-restricted modification system, resulting in low transformation efficiency. At the same time, Zhao Guangrong et al. did not investigate the effect of vgb expression in recombinant bacteria on the synthesis of target metabolites. influences

Method used

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  • Streptomyces diastatochromogenes engineering strain for producing toyocamycin, as well as construction method and application thereof
  • Streptomyces diastatochromogenes engineering strain for producing toyocamycin, as well as construction method and application thereof
  • Streptomyces diastatochromogenes engineering strain for producing toyocamycin, as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 : GFP gene gfp Construction of the genetic expression system of amylase Streptomyces chromogenes as a reporter gene:

[0033] Using plasmid pCAMBIA1302 as template, P gfp f Eco R I and P gfp R Hin dIII is the primer PCR obtained containing Eco R I and Hin dIII two enzyme cutting sites gfp Fragment, connected with pMD18-T Vector to construct cloning vector pMD18-T- gfp ( Eco R I+ Hin dIII). The cloning vector pMD18-T- gfp ( Eco R I+ Hind III ) into E. coli JM109 recipient bacteria were spread on the LB agar plate containing Amp, IPTG, and X-gal. After culturing overnight at 37 °C, many white colonies grew on the plate. Randomly pick white clones for enzyme digestion identification and send them to Shanghai Sangon Bioengineering Co., Ltd. for sequencing Eco R I and Hin dIII double digestion to obtain Eco R I and Hind III of the two enzyme cleavage sites gfp The fragment was ligated with pET28a that was digested with the...

Embodiment 2

[0039] Example 2 : GFP gene gfp Preliminary evaluation of the genetic expression system of amylase Streptomyces chromogenes as a reporter gene:

[0040] Pick the colony of Streptomyces amylase chromogenes grown on the plate containing 50 μg / mL apramycin and inoculate the CP medium, shake overnight at 28 °C and 180 r / min, and then inoculate inoculation amount of 1:50 on the Fill with 50 mL of fresh CP medium, culture to mid-logarithmic growth, dry cell weight (DCW) is about 0.3 g / L, collect mycelium by centrifugation at 6000 r / min, wash mycelium twice with PBS buffer Afterwards, resuspend in 30 mL PBS. Take 5-10 μL bacterial liquid smear, observe under Olympus BX50 fluorescence microscope, and take pictures with Color Video Camera of SONY 3CCD at the same time. Using wild bacteria as a control, select the excitation filter to be 485 nm and the emission filter to be 520 nm to determine the transcriptional activity of the promoter.

[0041] The results showed that the recom...

Embodiment 3

[0042] Example 3: Vitiligo hyaline hemoglobin gene vgb Expression in amylase Streptomyces chromogenes:

[0043] recombinant plasmid pIB139- vgb builds like Image 6 As shown, the steps are related to the recombinant plasmid pIB139- vgb The method of construction is similar.

[0044] like Figure 7 and Figure 8 Shown, respectively, recombinant plasmid pET28a- vgb and pIB139- vgb Enzyme digestion verification results. Recombinant plasmid pET28a- vgb through Eco R I and Hin DNA fragments of about 0.5kb and 5.3kb were obtained after dIII double digestion, respectively vgb The size of the gene fragment is the same as that of the plasmid pET28a. recombinant plasmid pIB139- vgb through Nde I and not I obtained a DNA fragment of about 0.6kb after double enzyme digestion, and vgb Gene fragments are of the same size. The above results prove that the recombinant plasmid pIB139- vgb builds correctly.

[0045] Extract plasmid pIB139- vgb , into amylase...

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Abstract

The invention discloses a streptomyces diastatochromogenes engineering strain for producing toyocamycin, as well as a construction method and application thereof. Vitreoscilla hemoglobingene (vgb) is integrated on a chromosome of the streptomyces diastatochromogenes engineering strain, so that the toyocamycin expression capability is higher than that of streptomyces diastatochromogenes 1628. The construction process is as follows: 1) constructing an expression vector pIB139-vgb; and 2) utilizing a conjugation method to integrate the expression vector into the chromosome of streptomyces diastatochromogenes to obtain the engineering strain. A promoter permE* on the pIB139 vector is utilized to start the expression of the vgb gene, and the conjugation method is utilized to integrate the specificity of the vector pIB139-vgb into the chromosome of the streptomyces diastatochromogenes 1628 to obtain the engineering strain with stable inheritance. Compared with an original strain, the yield of the recombinant toyocamycin is at least improved by 21.2%, and the concentration of the strain is improved by 11.6%.

Description

Technical field [0001] The present invention involves the use of transparent trembling hemoglobin genes to express the method of increasing the yield of abundance and puffedin in amylase chain mold, which belongs to the field of genetic engineering technology. Background technique [0002] Polycysiccin is a new type of nucleoside antibiotic, and the molecular formula is C 12 H 13 N 5 O 4 , Nuclear C 1 Connect the nitrogen -like purine -like purine -like purine ring, and the core structure is pyrine nitrilerine nucleoside analogs.The mechanism of action is mainly to affect the growth of the bacteria by inhibiting the transcription of microorganisms. Its biological activity research reports are mainly concentrated in the clinical medical field.Recently, some studies have found that Fumamycin has a good prevention and treatment effect on a variety of plant diseases. Long -term use will not cause environmental pollution, and it also has a certain regulatory effect on plant growth.The...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/31C12N15/76C12P19/40C12R1/525
Inventor 马正俞晓平刘金秀申屠旭萍边亚琳郝培应许益鹏
Owner CHINA JILIANG UNIV
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