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Recombinant streptomyces diastatochromogenes with reinforced adpA expression, construction method and application

A technology of Streptomyces chromogenes and amylase, applied in the field of genetic engineering, can solve complex problems, achieve the effect of enhancing the transcription level and increasing the production of toyocamycin

Active Publication Date: 2014-06-25
CHINA JILIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, 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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  • Recombinant streptomyces diastatochromogenes with reinforced adpA expression, construction method and application
  • Recombinant streptomyces diastatochromogenes with reinforced adpA expression, construction method and application
  • Recombinant streptomyces diastatochromogenes with reinforced adpA expression, construction method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0026] Example 1: Amplification of target gene and construction of recombinant amylase Streptomyces chromogenes

[0027] by S. diastatochromogenes 1628 chromosome genome as template, with PadpA F Nde I and PadpA R not I is a primer, PCR amplification obtains containing Nde I and not I two restriction sites adp A gene, connected with pMD18-T Vector to construct the cloning vector pMD18-T- adp A( Nde I + not I), the cloning vector pMD18-T- adp A( 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 two restriction sites adp A gene, connected with the same double-digested Streptomyces integrated shuttle expression vector pIB139 ( figure 1 ), the recombinant shuttle expression vector pIB139...

Embodiment 2

[0033] Embodiment 2: the expression of AdpA gene is to the synthetic key enzyme gene of toyocamycin toy Effect of F transcript levels

[0034] Recombinant amylase Streptomyces chromogenes 1628-ADPA 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. Detect the light absorbance at 260 to adjust the concentration of each RNA sample to make it consistent, and use the kit for one-step RT-PCR. 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 toyF gene can be amplified, and 16S rDNA was used as RT- Reference for PCR analysis. 16S rDNA F: 5’-CGATTACTAGCAACTCCGAC; 16S rDNA R: 5’-GGGGTGATGGGGACTCACAG. A 200 bp fragmen...

Embodiment 3

[0035] Example 3: Fermentation performance verification of amylase Streptomyces chromogenes original bacteria and recombinant bacteria

[0036] For the recombinant strain 1628-ADPA 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 adp The overexpression of A gene was verified to increase the production of toyocamycin. 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. After the fermentation, the content of toyocamycin in the fermentation liquid was determined. As shown in Table 1, the yield of the recombinant toyocamycin was higher than that of the original strain throughout the fermentation process, and the final yield of the recombinant toyocamycin reached 177.62 mg / L, which was about 31.6% higher than that of...

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Abstract

The invention discloses a recombinant streptomyces diastatochromogenes with reinforced adpA expression, a construction method and an application. A transcriptional regulation factor adpA at a secondary metabolism network center is excessively expressed, and the recombinant streptomyces diastatochromogenes has higher toyocamycin synthesis capacity compared with streptomyces diastatochromogenes 1628. The construction method comprises the following construction processes: (1) constructing an expression vector pIB139-adpA; and 2) merging the expression vector into a chromosome of the streptomyces diastatochromogenes by a conjugal transfer method so as to obtain engineering bacteria. The expression of adpA gene is started by utilizing a promoter permE* on the pIB139 vector, and the vector pIB139-adpA is specifically merged into the chromosome of the streptomyces diastatochromogenes 1628 by the conjugal transfer method so as to obtain the engineering bacteria with genetic stability. Compared with the original strain, the recombinant streptomyces diastatochromogenes has the advantages that the transcriptional level of a key enzyme gene toyF in the synthetic route of recombinant bacteria toyocamycin is obviously strengthened, and the yield of toyocamycin is improved by at least 31.6%. A foundation is laid for further improving the yield of toyocamycin and realizing industrial production as soon as possible.

Description

technical field [0001] The invention relates to enhancing the expression of adpA gene in amylase chromogenic streptomyces to increase the yield of toyocamycin, belonging 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 potential in the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/31C12N1/21C12N15/76C12P19/40C12R1/525
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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