Pyruvate response biosensor as well as construction method and application thereof

A biosensor, pyruvate technology, applied in the field of metabolic engineering, can solve the problems of dynamic control of the metabolic network of difficult strains, difficult to achieve fine control, etc., and achieve the effect of avoiding harmful effects and good specificity

Active Publication Date: 2020-09-22
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this sensor does not modify other necessary components, and it is difficult to achieve fine control of the expression of multiple metabolic pathways in the complex metabolic network, so it is difficult to be used for dynamic control of the metabolic network of strains

Method used

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  • Pyruvate response biosensor as well as construction method and application thereof
  • Pyruvate response biosensor as well as construction method and application thereof
  • Pyruvate response biosensor as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1: Construction of bacterial strain 168-PR

[0043] (1) Using the Escherichia coli K12 genome as a template, P43-pdhR-F and P43-pdhR-R as primers, amplify the pdhR fragment; using the Bacillus subtilis 168 genome as a template, pdhR-FF and pdhR-FR as primers, Amplify the upstream homology arm of pdhR; use pdhR-RF and pdhR-RR as primers to amplify the downstream homology arm of pdhR; artificially synthesize the lox71-spc-lox66-P43 cassette containing the spectinomycin gene and the P43 promoter ( The sequence is shown in SEQ ID NO.12); the pdhR fragment, the pdhR upstream homology arm, the pdhR downstream homology arm and the lox71-spc-lox66-P43 box were fused using fusion PCR technology to obtain the fusion gene fragment pdhRF-lox71-spc -lox66-P43-pdhR-pdhRR (refer to the Takara Primer STARMax DNA Polymeraser manual for the experimental method).

[0044] (2) Transform the fusion fragment obtained in step (1) into Bacillus subtilis 168 by electric shock, the e...

Embodiment 2

[0045] Example 2: Construction of pyruvate biosensor

[0046] Using the plasmid PHT01-P43-GFP preserved in the laboratory as a template (containing the P43 promoter, and the reporter gene egfp is placed downstream of P43), P43U-F / P43U-R, P43M-F / P43M-R and P43D-F / P43D-R was used as a primer, and the PdhR-binding sequence was inserted into the -35 region, -10 region and +1 site of the P43 promoter in the plasmid using inverse PCR technology to construct a hybrid promoter regulated by PdhR, thereby obtaining a hybrid promoter containing Recombinant plasmids PHT01-P43U-GFP, PHT01-P43M-GFP, and PHT01-P43D-GFP for pyruvate-responsive biosensors. Subsequently, the recombinant plasmids were transformed into Bacillus subtilis 168 and the recombinant strain 168-PR in Example 1 using the electric shock transformation technique, and then the fluorescence intensity of each recombinant plasmid in the corresponding strain was detected according to the detection method 2. The result is as ...

Embodiment 3

[0047] Example 3: Verification of the binding constant between P43D and pdhR in vitro

[0048] (1) Use the plasmid pET-28a preserved in the laboratory as a template, Z_PET28F and Z_PET28R as primers, and obtain linearized vector pET-28a by PCR amplification; use the Escherichia coli K12 genome as a template, 28apdhRF and 28apdhRR as primers, and perform PCR amplification A pdhR fragment containing homology arms was obtained. Then use NEB's Gibson Master Mix fused the pdhR fragment with the linearized vector pET-28a (refer to the product manual for specific steps) to obtain the recombinant plasmid pET-28a-pdhR. The recombinant plasmids were sent for sequencing, and the correctly sequenced plasmids were stored at -20°C.

[0049] (2) Transform the recombinant plasmid obtained in step (1) into E.coli BL21 competent (for the operation method, refer to the Basic Molecular Biology Manual), and obtain the recombinant strain E.coli BL21-pdhR containing the plasmid pET-28a-pdhR . T...

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Abstract

The invention discloses a pyruvate response biosensor as well as a construction method and application thereof. The pyruvate response biosensor with different dynamic ranges is successfully constructed in the invention by optimizing a PdhR binding sequence inserted into a P43 promoter and optimizing an insertion site, wherein the minimum dynamic range is increased by 0.6 times, and the maximum dynamic range is increased by 30.7 times. The biosensor can be used for finely controlling the expression quantity of each gene in cells. Pyruvic acid is a key central carbon metabolite in cells, so thatthe biosensors can dynamically regulate and control the expression levels of intracellular genes according to the change of the content of pyruvic acid in the cells, thereby realizing dynamic controlof intracellular metabolic flux. The pyruvate biosensor obtained by the invention has good specificity, and in addition, the response range of the pyruvate biosensor to pyruvic acid is 10-35nmol / g DCW.

Description

technical field [0001] The invention relates to a pyruvate-responsive biosensor and its construction method and application, belonging to the technical field of metabolic engineering. Background technique [0002] The main purpose of metabolic engineering is to use microorganisms as hosts and biomass as raw materials to efficiently synthesize required chemicals, such as drugs, materials, biofuels, fine chemicals and functional nutrients. However, traditional metabolic engineering techniques (overexpression of genes related to product synthesis pathways, knockout of competing pathways, and optimization of cofactors, etc.) often lead to problems such as intracellular metabolic imbalance, low raw material utilization, and accumulation of toxic metabolites. , making it difficult to maximize compound yield and production intensity. In addition, although the traditional mutation breeding method can quickly obtain a large number of mutant strains, it is difficult to quickly obtain...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/31
CPCC12N15/113C07K14/32C12N2830/002C12N15/75C12R2001/125
Inventor 刘龙陈坚堵国成李江华吕雪芹徐显皓
Owner JIANGNAN UNIV
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