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High-throughput screening method based on biosensor and application of high-throughput screening method in multi-gene metabolic pathway optimization

A screening method and sensor technology, applied in the field of biochemistry, can solve problems such as the lack of high-throughput screening methods for glycolic acid production strains, and achieve the effects of avoiding fluorescence quenching, avoiding difficult identification, and expanding screening throughput

Pending Publication Date: 2021-07-02
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Lack of high-throughput screening methods for glycolic acid-producing strains is a major challenge to systematically optimize their synthetic pathways

Method used

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  • High-throughput screening method based on biosensor and application of high-throughput screening method in multi-gene metabolic pathway optimization
  • High-throughput screening method based on biosensor and application of high-throughput screening method in multi-gene metabolic pathway optimization
  • High-throughput screening method based on biosensor and application of high-throughput screening method in multi-gene metabolic pathway optimization

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Design and Construction of Glycolic Acid Sensor

[0048] Such as figure 1 As shown in A, pGBS-P ffS - sfGFP sensor includes promoter P glcD , sfGFP protein, promoter P ffS , glcC gene; The promoter P glcD To initiate expression of sfGFP, P glcD There is a cis-response element enhancing factor UAS on it; the P glcD contains the binding site of GlcC-glycolate allosteric protein; the downstream of glcC is the kanamycin resistance gene and the thermosensitive replicon Ori101; the P glcD Located downstream of the replicon Ori101, the glcC upstream sets the promoter P ffS . (the content is the patent of CN110684792A with publication number).

[0049] Biosensor pGBS-P ffS -tetA builds like figure 1 C, pGBS-P ffS -tetA sensor will pGBS-P ffS - The reporter gene sfGFP protein in sfGFP was replaced by the tetracycline resistance gene tetA, and the other structures were unchanged. in pGBS-P ffS Amplified linearized vector P with primers GBS-F / GBS-R on the b...

Embodiment 2

[0052] Example 2 Response of glycolic acid sensor to glycolic acid

[0053] In order to detect the response performance of the glycolic acid sensor, the expression of the reporter gene was induced by exogenous addition of different concentrations of glycolic acid.

[0054] After pGBS-PffS-sfgfp was introduced into the host strain MG1655ΔglcC, a single colony was picked and inoculated into a kanamycin-resistant liquid LB medium, and cultivated overnight at 30°C at 250 rpm to prepare a seed solution. The seed solution was divided into initial OD 600 =0.05 of the inoculum was inoculated into the M9 medium, which contained a glucose concentration of 4g / L. After culturing for 7-8h, wait for OD 600 The expression of green fluorescent protein was induced by adding different concentrations of glycolic acid to about 0.6. Continue to culture under the same conditions for 6 hours, take a sample of 50 μL and dilute it 3 times with PBS buffer solution, then measure the fluorescence valu...

Embodiment 3

[0056] Construction of embodiment 3 pathway library

[0057] In glycolic acid production, there are three key pathway genes, ycdW, aceA and gltA, which are controlled by inducible promoters ptrc and T7 promoters. Replace the Trc and T7 promoters with a series of promoters with gradient strengths to fine-tune metabolic pathways and avoid the use of IPTG. 22 PUTRs with gradient strength (promoter and 5'UTR complex) were amplified and randomly connected to the upstream of the three pathway genes respectively, and the constructed plasmid library was co-transformed into the glycolic acid engineering strain Mgly6(pGBS -P ffS -tetA), the strain was named Mgly6-TP6101, generating a final library with a theoretical diversity of 10648. The specific construction steps are as follows image 3 Shown:

[0058]Using the plasmid pJUN-YA containing the gene ycdW as a template, the linearized vector ptrc-ycdWaceA was amplified with primers PycdW-F / PycdW-R, and the plasmid pJUN-5 containing ...

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Abstract

The invention discloses a high-throughput screening method based on a biosensor and application of the high-throughput screening method in multi-gene metabolic pathway optimization, and belongs to the technical field of high-throughput screening. According to the invention, an efficient high-throughput screening method is constructed by combining two biosensors of specific response glycollic acid, high-throughput screening is carried out on a multi-gene pathway library of metabolite micromolecule glycollic acid, and optimization of glycollic acid multi-gene metabolic pathways is realized in a short time; high-yield strains are selected based on plate resistance, so that the screening flux can be greatly expanded, and the high-yield strains are simply and rapidly enriched; the concentration of glycollic acid in the fermentation liquor is rapidly detected by combining a 96-well plate fluorescence determination method; according to the method, repeated elimination of plasmids can be realized by combining a glycollic acid sensor and a temperature-sensitive replicon, and iterative high-throughput screening is facilitated; and through the improvement, the screening sensitivity of the target high-yield strain is effectively improved, and the enrichment efficiency of the high-yield strain is improved.

Description

technical field [0001] The invention relates to a biosensor-based high-throughput screening method and its application in the optimization of multi-gene metabolic pathways, belonging to the technical field of biochemistry. Background technique [0002] Glycolic acid is a kind of α-glycolic acid with multiple uses in industry, and has been widely used in cosmetics, chemical cleaning and degradable materials and other fields. In recent years, with the continuous development of metabolic engineering and synthetic biology, the production of glycolic acid by total biosynthesis has attracted extensive attention due to its mild reaction, high product purity, easy availability of raw materials, and green and sustainable advantages. Previous studies have strengthened the metabolic flux of glycolic acid through metabolic engineering, and the production of glycolic acid has been significantly improved. Therefore, some expensive inducer-dependent strong promoters (such as T7, Trc promot...

Claims

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Application Information

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IPC IPC(8): C12Q1/04C12N15/65G01N21/64C12P7/42
CPCC12Q1/04C12N15/65G01N21/6486C12P7/42C12N2830/001C12N2820/002
Inventor 邓禹周胜虎徐淑敏毛银赵磊陆雨洁肖潇王心仪
Owner JIANGNAN UNIV
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