Transcription factor NCgl0581 mutant and use thereof in L-serine detection

Patent metadata

Field	Value
Publication number	US-12448422-B2
Application number	US-202418957819-A
Country	US
Kind code	B2
Filing date	Nov 24, 2024
Priority date	Sep 6, 2023
Publication date	Oct 21, 2025
Grant date	Oct 21, 2025

Abstract

The present invention provides a transcription factor NCgl0581 mutant and use thereof in L-serine detection, belonging to the technical field of biological detection. According to the present invention, key binding sites of the transcription factor NCgl0581 with L-serine are obtained through molecular docking, and further model analysis and virtual screening are carried out to obtain an L-serine biosensor mutant NCgl0581E136P. Experiments show that the biosensor constructed based on the mutant NCgl0581E136P can respond to 25 mM serine, which is half of the response concentration before the mutation. The present invention reduces the detection limit of the biosensor through mutation, lays a foundation for the detection of low-concentration L-serine, and has good application value and prospect.

First claim

What is claimed is:

1. A transcription factor NCgl0581 mutant, wherein the mutant is obtained by mutating an 136th glutamic acid of an amino acid sequence as shown in SEQ ID NO.1 into proline.

2. A nucleic acid encoding the transcription factor NCgl0581 mutant according to claim 1 .

3. An expression vector carrying the nucleic acid according to claim 2 .

4. An L-serine detection product comprising a transcription factor NCgl0581 mutant, wherein the mutant is obtained by mutating the 136 th glutamic acid of an amino acid sequence as shown in SEQ ID NO. 1 into proline, wherein the L-serine detection product further comprises a biosensor.

5. The L-serine detection product according to claim 4 , wherein the biosensor comprises a coding gene of the transcription factor NCgl0581 mutant, a promoter for initiating expression of the coding gene of the transcription factor NCgl0581 mutant, a promoter P SerE and a reporter gene whose expression is activated by the promoter P SerE .

6. A method for detecting L-serine, comprising the steps of incubating host cells containing the biosensor of claim 5 with a sample to be tested, and performing signal detection.

7. The method according to claim 6 , further comprising the steps of:
S1, incubating a plurality of first samples containing L-serine with different concentrations with the host cells containing the biosensor, and detecting signal intensity of a reporter gene after the incubation is completed;
S2, establishing a relationship curve between signals of the reporter gene and concentrations of the L-serine in the first samples; and
S3, repeating the step S1 but replace the first samples with a second sample to be tested, and substituting a result into the relationship curve in S2 to calculate the concentration of the L-serine in the second sample,
wherein the concentrations of the L-serine in the first samples are known, and the concentration of the L-serine in the second sample is unknown.

Assignees

• Jitri Future Food Tech Institute Co Ltd
• Univ Jiangnan

Inventors

• Zhang Xiaomei
• Huang Yamin
• Gao Yujie
• Xu Guoqiang
• Li Hui
• Shi Jinsong
• Xu Zhenghong

Classifications

• C12Q1/6897

  involving reporter genes operably linked to promoters · CPC title

• G01N33/68

  involving proteins, peptides or amino acids {(involving lipoproteins G01N33/92)} · CPC title

• C07K14/4705 Primary

  stimulating, promoting or activating activity · CPC title