Gntr family transcriptional repressor mutants, mutant genes and their role in the production of vitamin b 2 application in

A technology of transcriptional repressors and mutants, which is applied in the field of preparation of vitamin B2, can solve the problems that there are no GntR family transcriptional repressor mutant genes, etc., and achieve great application value and the effect of improving ability

Active Publication Date: 2022-05-10
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 transcriptional repressor mutants, mutant genes and their role in the production of vitamin b <sub>2</sub> application in
  • Gntr family transcriptional repressor mutants, mutant genes and their role in the production of vitamin b <sub>2</sub> application in
  • Gntr family transcriptional repressor mutants, mutant genes and their role in the production of vitamin b <sub>2</sub> application in

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 fusion PCR, get the assembly fragment UCR-ytrA m , was detected correctly by nucleic acid elec...

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 Under sterile conditions, the LB solid plate containing 25 mg / L erythromycin was streaked with an inoculation needle, and cultured 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 s...

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Abstract

The invention discloses a GntR family transcription inhibitor mutant, a mutant gene and an application thereof in preparing vitamin B2. Wherein the amino acid sequence of the mutant has the following mutations relative to the sequence shown in SEQ ID No.3: the 89th amino acid is replaced by M. The 89th amino acid coding nucleotide in the GntR family transcription repressor gene on the Bacillus subtilis chromosome is subjected to site-directed mutation to encode the amino acid M, and the genetic engineering bacteria obtained have a relatively improved ability to produce vitamin B2 , and is beneficial to the growth of bacteria, so it has great application and promotion value.

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 Patents(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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