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L-cysteine biosensor and application thereof

A biosensor, cysteine ​​technology, applied in the direction of application, microbes, and microbe-based methods, etc., can solve the problems of rapid screening and isolation of L-cysteine ​​high-yielding strains

Active Publication Date: 2022-01-28
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, generally speaking, the rapid screening and isolation of L-cysteine ​​high-yielding strains are still relatively limited, so it is urgent to construct a similar high-efficiency biosensor to solve the bottleneck problem of L-cysteine ​​strain breeding

Method used

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  • L-cysteine biosensor and application thereof
  • L-cysteine biosensor and application thereof
  • L-cysteine biosensor and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Embodiment 1: Construction of L-cysteine ​​biosensor plasmid

[0020] In this example, the biological elements of each component in the L-cysteine ​​biosensor were amplified by PCR method, and assembled by one-step efficient seamless cloning (ClonExpress®II One Step Cloning Kit, Vazyme Biotech, China) and other molecules Technology Connect the fragments in a certain order to complete the construction of the L-cysteine ​​biosensor plasmid.

[0021] In Pantoea pineapple, CcdR protein is the first discovered L-cysteine ​​transcriptional regulator, which can regulate the expression of the downstream ccdA gene. After the exogenous addition of 0.1 mmol / L L-cysteine, the transcription of ccdA levels can be increased hundreds of times (Takumi K, et al. Bacterial cysteine-inducible cysteine ​​resistance systems. JBacteriol. 2016;198:1384-1392.). As a transcription regulator that interacts with L-cysteine, CcdR has a high response speed and sensitivity. When there is a certain...

Embodiment 2

[0031] Example 2: Transformation and optimization of L-cysteine ​​biosensor

[0032] CcdR is a transcriptional regulator that interacts with L-cysteine, and its regulatory activity is closely related to the response speed and sensitivity of biosensors. Through directed evolution of CcdR protein, screening for better CcdR protein mutants and enhancing binding specificity and affinity will help to further improve and enhance the sensitivity of L-cysteine ​​biosensors.

[0033] Pantoea pineapple P. ananatis The AJ13355 genome was used as a template, and the CcdR coding gene mutation library was amplified by error-prone PCR random mutation method using primers F4 and R4. By adding a certain concentration of magnesium ions and manganese ions to the PCR reaction system, the fidelity of the PCR amplification process is further reduced, and the obtained coding genes are controlled to contain 1 to 3 point mutations. Using the L-cysteine ​​biosensor plasmid Pcys-Bio obtained in Exam...

Embodiment 3

[0043] Example 3: Screening of L-cysteine ​​high-yielding strains based on improved biosensors

[0044]As an inconspicuous small molecule compound, L-cysteine ​​is difficult to detect in cells, and it is difficult to develop high-throughput screening methods. Based on the improved biosensor that can sensitively respond to changes in intracellular L-cysteine ​​concentration and output as a fluorescent signal, it can be quickly sorted at the fluorescent level by coupling high-throughput screening equipment such as flow cytometry. It greatly improves the speed of high-yielding strain breeding and the potential of efficiently screening target strains from diversified mutant libraries.

[0045] Such as image 3 Shown is a schematic flow chart of the high-throughput screening of L-cysteine. The specific steps are: select the CYS-2 strain with basic L-cysteine ​​fermentation ability containing the Pcys-Bio-V94I reporter system (knockout cystine degradation pathway genes wxya and ...

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Abstract

The invention provides construction and application of an L-cysteine biosensor. The biosensor contains a CcdR coding gene, a control sequence PccdR promoter of the CcdR coding gene, a reporter gene and a control sequence PccdA promoter of the reporter gene, wherein a CcdR transcriptional regulatory factor can be specifically combined with a PccdA promoter region to induce and activate expression of reporter protein. The biosensor shows an excellent linear response relationship to L-cysteine, can be used for efficient breeding of L-cysteine high-yield strains or directed evolution of L-cysteine synthetic pathway key rate-limiting enzyme by being combined with a high-throughput screening system and the like, and has the advantages of being simple to operate, short in detection period, high in detection efficiency, high in sensitivity and the like.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a specific biosensor for L-cysteine ​​and its application. Background technique [0002] L-cysteine ​​is a sulfur-containing amino acid with important physiological functions, and has a wide range of uses in the fields of food, medicine, cosmetics and feed. With the continuous development of the terminal use of L-cysteine, the demand for cysteine ​​in the global market is increasing rapidly, and the market prospect is very bright. According to media reports, the total global production of L-cysteine ​​in 2016 was between 14,000 and 15,000 tons, and the market size of cysteine ​​and its downstream derivatives has approached or exceeded US$400 billion. my country's L-cysteine ​​production accounts for about 2 / 3 of the world's total production, and the annual export volume remains above 80%, occupying an important position in the highly competitive international market. A...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/77C12N15/31C12N15/65C12N1/21C07K14/195G01N15/14C12R1/19
CPCC12N15/77C12N15/65C07K14/195G01N15/14
Inventor 刘君高金山魏亮徐宁
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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