A dynamic regulation system controlled by combination of promoters

A technology of promoters and genetically engineered bacteria, applied in the field of bioengineering, can solve problems such as growth defects, and achieve the effects of low strain growth load, fewer system components, and simple design

Active Publication Date: 2020-09-04
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Directly blocking the synthesis of shikimate-3-phosphate will cause growth defects of the strain in inorganic salt medium

Method used

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  • A dynamic regulation system controlled by combination of promoters
  • A dynamic regulation system controlled by combination of promoters
  • A dynamic regulation system controlled by combination of promoters

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Evaluation of promoter activity

[0054] After the growth phase-associated promoter gene was synthesized, B0034RBS and reporter gene GFP were added by fusion PCR. The above PCR product was recovered and ligated with the vector PJ01 plasmid to construct the recombinant plasmid PJ01-GPP-GFP.

[0055] After the gene synthesis of the stable phase-associated promoter, B0034RBS and reporter gene GFP were added by fusion PCR. The above PCR product was recovered and connected to the vector pTet-1 plasmid to construct the recombinant plasmid P SPP -GFP.

[0056] The obtained recombinant plasmid PJ01-GPP-GFP was introduced into the competent cell E.coli JM109 to obtain a strain containing a growth-associated promoter evaluation plasmid.

[0057] The obtained recombinant plasmid P SPP -GFP was introduced into the competent cell E.coli JM109 to obtain a strain containing a stable phase evaluation plasmid.

[0058] In LB medium, the above strains were subjected to con...

Embodiment 2

[0059] Example 2 Assembly and detection of dynamic regulatory gene circuits

[0060] In order to construct a dynamic regulatory gene circuit, the protease TEV gene fragment was used to replace the above P SPP -Reporter gene GFP in GFP, to obtain plasmid P SPP -TEV plasmid. At the same time, the N-terminal modification of the reporter gene GFP of the recombinant plasmid PJ01-GPP-GFP was carried out, and the TEV protease recognition cleavage site and phenylalanine degradant were added to obtain the plasmid PJ01-GPP-(teF)GFP. Plasmid P SPP -TEV and PJ01-GPP-(teF)GFP were co-transformed into JM109, screened using ampicillin and chloramphenicol dual resistance plates, and the transformants were verified by PCR, and finally obtained the dynamic regulation line detection strain containing the above double plasmid .

[0061] The above-mentioned detection strains were cultured in LB medium, and the continuous fluorescence measurement was performed using a SpectraMax M3 microplate m...

Embodiment 3

[0062] The construction of embodiment 3 shikimic acid production strains

[0063] E.coli MG1655 was selected as the chassis engineering bacteria, shikimate kinase I and II (aroK, aroL) of the host bacteria were knocked out, and the PTS system of the host bacteria was replaced by the glucose facilitation protein Zmglf derived from Zymomonas mobilis. The new strain was named S4, because the strain blocked the shikimic acid pathway, it could not grow in the NBS inorganic salt medium, let alone accumulate the product shikimic acid.

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Abstract

The invention discloses a promoter combination controlling dynamic regulation and control system and discloses the application of the promoter combination controlling dynamic regulation and control system and application thereof to the production of shikimic acid, and belongs to the technical field of bioengineering. Two types of promoters with completely different transcription characteristics are screened through a molecular biology method, and comprise a growth period correlated promoter and a stable period promoter. According to the property of specifically identifying cut specific short peptide of proteinase, a protein abundance regulation and control system, which does not need manual control and exogenous addition of an inducing agent, is designed and constructed. A dynamic regulation and control gene path targeting shikimate kinase is introduced into engineering escherichia coli, so that the capability of producing shikimic acid without exogenously adding aromatic amino acid and the inducing agent in an inorganic salt culture medium is realized. According to the dynamic regulation and control system, the yield of the produced shikimic acid can reach 21.2 g / L and the conversion rate reaches 0.24 g / g glucose.

Description

technical field [0001] The invention relates to a dynamic regulation system controlled by combination of promoters, in particular to a dynamic regulation system controlled by combination of promoters and its application in shikimic acid production, belonging to the technical field of bioengineering. Background technique [0002] The dynamic control system is an emerging metabolic flow control method in the field of metabolic engineering. Its main feature different from the static control is that during the fermentation process, the engineered strain will make changes according to the fermentation time, physiological state, intracellular metabolite concentration, and extracellular environment changes. The corresponding adjustment of enzyme activity can affect the distribution of metabolic flux and improve the production capacity of products. The dynamic control system has the advantages of no need for artificial regulation in the fermentation process, and no need for exogenou...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12P7/42C12R1/19
CPCC12N9/1205C12N9/506C12P7/42C12Y207/01071
Inventor 刘立明高聪侯建屾叶超陈修来罗秋玲刘佳
Owner JIANGNAN UNIV
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