Frr expression reinforced recombined streptomyces diastatochromogenes, as well as construction method and use

A technology for producing Streptomyces chromogenes and amylase, which is applied in the field of genetic engineering, can solve the problems of frameshift mutation, complex and complicated operation of gene fusion fragment process, etc., and achieve the effects of increasing yield, enhancing transcription level, and shortening fermentation cycle.

Inactive Publication Date: 2013-09-11
CHINA JILIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Li Lili et al. used SOE-PCR (Splicing by overlap extension PCR) technology to amplify the erythromycin resistance gene promoter PermE and frr structural gene fusion fragment, inserting the strand The mold-integrated vector pSET152, and the frr gene was integrated into the abamectin production strain by conjugative transfer method, the production of avermectin was improved, but the process of obtaining the gene fusion fragment was operated Complex, prone to frameshift mutations
In addition, this strategy has only been successful on a few specific strains and specific target products
For the amylase Streptomyces chromogenes, complex factors are involved from the establishment of the expression system to the improvement of the target product, and there is no report yet

Method used

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  • Frr expression reinforced recombined streptomyces diastatochromogenes, as well as construction method and use
  • Frr expression reinforced recombined streptomyces diastatochromogenes, as well as construction method and use
  • Frr expression reinforced recombined streptomyces diastatochromogenes, as well as construction method and use

Examples

Experimental program
Comparison scheme
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 a template, with Pfrr F Nde I and Pfrr R not I is a primer, PCR amplification obtains containing Nde I and not I The frr gene with two restriction sites was connected with pMD18-T Vector to construct the cloning vector pMD18-T-frr ( Nde I + not I), the cloning vector pMD18-T-frr ( Nde I + not I) Transformed into recipient Escherichia coli, smeared on LB agar plate containing Amp, cultured overnight at 37 ℃, 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 The frr gene of the two restriction sites is connected with the Streptomyces integrated shuttle expression vector pIB139 of the same double digestion, and the recombinant shuttle expression v...

Embodiment 2

[0036] Example 2: Frr The effect of gene expression on the transcription level of key enzyme gene toyF in toyocamycin synthesis

[0037] Recombinant amylase Streptomyces chromogenes 1628-FRR and the original strain were cultured in CP medium for 48 hours, and the cells were harvested, and total RNA was extracted and DNA was removed using kits. The purity and quality of RNA samples were determined by measuring the ratio of absorbance at 260 and 280 nm and agarose gel electrophoresis. Measure the absorbance at 260 to adjust the concentration of each RNA sample to make it consistent. RT-PCR using the kit one-step method. Gene toy F is one of the key enzyme genes in the biosynthesis of toyF, using primers: toyF F: 5'-CTGTCGCTGGAGCTGGTGCG; toyF R: 5'-CAGCGACGAGGGCGCGGCGG. A 400 bp fragment of the toyF gene can be amplified. Compared with the original strain 1628, the recombinant strain during fermentation frr The overexpression of the gene significantly enhanced the transcr...

Embodiment 3

[0038] Example 3: Verification of Fermentation Performance of Amylase Streptomyces Chromogenes Original Strain and Recombinant Strain

[0039] For the recombinant strain 1628-FRR and the original strain S. diastatochromogenes 1628 conducted a 250 mL shake flask fermentation experiment, and studied the amylase chromogenic Streptomyces from the perspective of fermentation frr The effect of overexpression of the gene on the improvement of the production of toyocamycin was verified. The speed of the reciprocating shaker was 200 r / min, 28°C, and the control group was the origin strain of Streptomyces chromogenes amylase. After the fermentation, the content of toyocamycin in the fermentation liquid was determined. During the entire fermentation process, the yield of the recombinant toyocamycin was higher than that of the original bacteria, and the final yield of the recombinant toyocamycin reached 197.46 mg / L, which was about 46.3% higher than that of the original bacteria (Ta...

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Abstract

The invention discloses an frr expression reinforced recombined streptomyces diastatochromogenes, as well as a construction method and use. The recombined streptomyces diastatochromogenes excessively expresses ribosome cycle factor frr and has higher toyocamycin synthesis capability than streptomyces diastatochromogenes 1628. The construction process is as follows: 1) constructing an expression vector pIB139-frr; and 2) utilizing a conjugational transfer method to integrate the expression vector with the chromosome of the streptomyces diastatochromogenes so as to obtain the engineering bacteria. A promoter permoE* on the pIB139 vector is utilized to start expression of frr gene; the vector pIB139-frr is specifically integrated with the chromosome of the streptomyces diastatochromogenes 1628 by the conjugational transfer method so as to obtain engineering bacteria with genetic stability. Compared with the original strain, the engineering bacteria obviously improves the transcriptional level of the key enzyme gene toyF in the recombined toyocamycin synthesis route, increases the yield of the toyocamycin by at least 43.6%, and shortens the fermentation period by 12h.

Description

technical field [0001] The present invention involves enhancing the frr The expression of gene in streptomyces chromogenes amylase improves the yield of toyocamycin, which 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 potent...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/76C12P19/28C12R1/525
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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