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GntR family transcription inhibitor mutant, mutant gene and application of the mutant gene to preparation of vitamin B2

A technology of transcription inhibitor and mutant, which is applied in the application field of preparing vitamin B2, can solve the problem that there is no mutant gene of GntR family transcription inhibitor, and achieve the effect of great application value and improving ability.

Active Publication Date: 2021-01-15
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

At present, there is no report that the GntR family transcriptional repressor mutant gene ytrA is used for vitamin B2 synthesis

Method used

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  • GntR family transcription inhibitor mutant, mutant gene and application of the mutant gene to preparation of vitamin B2
  • GntR family transcription inhibitor mutant, mutant gene and application of the mutant gene to preparation of vitamin B2
  • GntR family transcription inhibitor mutant, mutant gene and application of the mutant gene to preparation of vitamin B2

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Strains CGMCC NO.16132 and BS168 ribC m Perform comparative genomic analysis

[0030] strain BS168 ribC m (Refer to Chinese patent application 201910604170.3 for the specific construction process) The genome is used as the reference genome, and the strain CGMCC NO.16132 is sent to Jinweizhi Biotechnology Co., Ltd. for whole-genome resequencing and differential analysis, in order to find out the ability to produce vitamin B2. Gene. Through sequence comparison analysis, it was found that a large number of mutations occurred in the strain CGMCC NO.16132, which contained a total of 338 mutation sites: after mutation data statistics, a total of 72 genes were involved in the mutation. Mutations in the coding region involved a total of 59 genes, including 8 synonymous mutations, 41 non-synonymous mutations, 2 nonsense mutations, 5 frameshift mutations, and 3 non-frameshift mutations; mutations in non-coding regions A total of 13 genes were involved.

[0031] Rem...

Embodiment 2

[0032] Embodiment 2: construct the bacterial strain containing ytrA mutant

[0033] Using Bacillus subtilis 168 chromosome as template, primer UPytrA m -F, UPytrA m -R (with DR) to amplify UPytrA with adapter and point mutation m (DR-containing) fragment, use primers araR-F, araR-R (containing DR) to amplify the araR (containing DR) fragment with adapter; use pC194 plasmid as template, use primers cat-F, cat-R to amplify the fragment with adapter The cat fragment of the bacterial strain CGMCC NO.16132 is used as a template, and the downstream homology arm fragment DNytrA is amplified with primers DNytrA-F and DNytrA-R, wherein the nucleotide sequence of the mutated ytrA is as SEQ ID No.2 As shown, the encoded amino acid sequence is shown in SEQID No.4.

[0034] with UPytrA m Fragment, cat fragment, araR fragment and DNytrA fragment as templates, use primer UPytrA m -F, DNytrA-R for fusion PCR to obtain the assembled fragment UCR-ytrA m , was detected correctly by nucleic...

Embodiment 3

[0042] Example 3: Evaluation of B vitamins in different strains 2 production capacity

[0043] 1. Strain culture conditions:

[0044] Will start strain BS168 ribC m , and engineering strain BS168 ytrA m Streak the LB solid plate containing 25mg / L erythromycin with an inoculation needle under sterile conditions, and incubate it upside down in a 37°C incubator for 24 hours to obtain freshly activated single colonies. Pick a single colony with an inoculation needle and streak the LB solid slope containing 25 mg / L erythromycin, and culture it in a 37°C incubator for 48 hours. Scrape 1 / 3 of the bacterial lawn on the inclined surface and inoculate it into a 500mL baffled Erlenmeyer flask containing 70mL of fermentation medium (3 parallels for each strain of bacteria), shake culture at 37°C and 200rpm for 41h, then take the fermentation broth to measure OD600 and vitamin B 2 Yield.

[0045] 2. OD600 and vitamin B of different strains 2 Yield Comparison

[0046] with the starti...

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Abstract

The invention discloses a GntR family transcription inhibitor mutant, a mutant gene and an application of the mutant gene to preparation of vitamin B2. The amino acid sequence of the mutant has the following mutation relative to the sequence shown in SEQ ID No.3: the 89th amino acid is replaced with M. The 89th amino acid encoding nucleotide in the GntR family transcription inhibitor gene on the chromosome of bacillus subtilis is subjected to site-specific mutagenesis to encode the amino acid M. The vitamin B2 production capacity of the obtained genetically engineered bacterium is greatly improved, and growth of the bacterium is facilitated, so that high application and popularization values are achieved.

Description

Technical field: [0001] The invention belongs to the field of biotechnology, and in particular relates to a mutant of a GntR family transcriptional repressor, a genetically engineered bacterium thereof, and an application in preparing vitamin B2. Background technique: [0002] Riboflavin (Riboflavin) also known as vitamin B 2 , the molecular formula is C 17 h 20 o 6 N 4 , is a water-soluble vitamin in the vitamin B family. It exists in two forms of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) in the organism, and acts as some important redox enzymes in the body. The coenzymes involved in redox reactions play a role in delivering hydrogen. The lack of riboflavin will lead to metabolic disorders in organisms, but neither humans nor animals can synthesize it by themselves and can only ingest it from food. Therefore, the production of riboflavin has a very broad market in the food, feed and pharmaceutical industries. [0003] At present, the industrial...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/32C12N15/31C12N1/21C12P25/00C12R1/125
CPCC07K14/32C12P25/00
Inventor 张大伟孙宜文刘川
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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