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Recombinant streptomyces diastatochromogenes with reinforced toyF expression, construction method and uses thereof

A technology for producing Streptomyces chromogenes and amylase, which is applied in the field of genetic engineering to achieve the effect of increasing yield

Active Publication Date: 2014-07-23
CHINA JILIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, using metabolic engineering technology to increase the metabolic flux of toyocamycin to further increase the yield of toyocamycin has not been reported because of complex issues such as the establishment of an expression system.

Method used

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  • Recombinant streptomyces diastatochromogenes with reinforced toyF expression, construction method and uses thereof
  • Recombinant streptomyces diastatochromogenes with reinforced toyF expression, construction method and uses thereof
  • Recombinant streptomyces diastatochromogenes with reinforced toyF expression, construction method and uses thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Example 1: Amplification of Target Gene and Construction of Recombinant Amylase Streptomyces Chromogenes

[0030] by S. diastatochromogenes 1628 chromosome genome as template, PtoyF F Nde I and Ptoy F R not I is a primer, PCR amplification obtains containing Nde I and not I The toyF gene with two restriction sites was connected with pMD18-T Vector to construct the cloning vector pMD18-T-toyF ( Nde I + not I), the cloning vector pMD18-T-toyF ( Nde I + not I) Transformed into recipient Escherichia coli, smeared on LB agar plate containing ampicillin resistance, cultured overnight at 37°C, randomly picked positive transformants and sent them to Shanghai Sangong for sequencing and sequence analysis. Nde I and not I double digestion to obtain containing Nde I and not I connect the toyF gene with two restriction sites to the Streptomyces integrated shuttle expression vector pIB139 with the same double digestion, and obtain the recombinant sh...

Embodiment 2

[0036] Example 2: Verification of Fermentation Performance of Amylase Streptomyces Chromogenes Original Strain and Recombinant Strain

[0037] Compared with the original strain, the adenosine succinate lyase activity of the recombinant strain was increased by 0.9 times, and the recombinant strain 1628-TOYF and the original strain S. diastatochromogenes 1628 conducted a 250 mL shake flask fermentation experiment to verify the effect of the enhancement of adenosuccinate lyase activity on the production of toyocamycin in Streptomyces chromogenes amylase from the perspective of fermentation. The rotating speed of the reciprocating shaker was 200 r / min, 28°C, and fermented for 96 hours. The control group was the origin strain of Streptomyces chromogenes amylase. As shown in Table 1, the yield of the recombinant strain toyocamycin was higher than that of the original strain, and the final yield of the recombinant strain toyocamycin reached 162.64 mg / L, which was about 20.5% high...

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Abstract

The invention discloses a recombinant amylase streptomyces chromogenes with enhanced expression of toyF, its construction method and application. It overexpresses the key enzyme of biosynthesis toyocamycin-adenosuccinate lyase encoding gene toyF, and has higher toyocamycin synthesis ability than amylase Streptomyces diastatochromogenes (Streptomyces diastatochromogenes) 1628. The construction process is as follows: 1) Construct the expression vector pIB139-toyF; 2) Use the conjugative transfer method to integrate the expression vector into the chromosome of Streptomyces chromogenes amylase to obtain the engineering bacteria. The promoter permE* on the pIB139 vector was used to promote the expression of the toyF gene, and the vector pIB139-toyF was specifically integrated into the chromosome of Streptomyces diastatochromogenes 1628 by conjugative transfer method, and genetically stable engineering bacteria were obtained. Compared with the original strain, the enzyme activity of adenosine succinate lyase of the recombinant strain was increased by 0.9 times, and the yield of toyocamycin was increased by at least 20.5%.

Description

technical field [0001] The invention relates to improving the yield of toyocamycin by strengthening the expression of the toyF gene in the amylase streptomyces chromogenes, and belongs to the technical field of genetic engineering. Background technique [0002] Toyocamycin is a new type of nucleoside antibiotics with the molecular formula C 12 h 13 N 5 o 4 , Ribose C 1 The deazapurine ring similar to guanine is connected, and the core structure is a pyrropyrimidine nucleoside analogue. The mechanism of action is mainly to affect the growth of bacteria by inhibiting the transcription of microorganisms, and the research reports on its biological activity are mainly concentrated in the field of clinical medicine. Recent studies have found that toyocamycin has a good control 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. Therefore, toyocamycin has great application po...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/60C12N15/76C12P19/40C12R1/525
Inventor 马正俞晓平陶立彬申屠旭萍边亚琳郝培应许益鹏
Owner CHINA JILIANG UNIV
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