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Ribonucleotide reductase transcription inhibitor mutant, mutant gene and its application in the preparation of vitamin b2

A transcription inhibitor and ribonucleotide technology, applied in the biological field, can solve the problem that there is no mutation base of ribonucleotide reductase transcription inhibitor, and achieve the effect of great application value and ability improvement.

Active Publication Date: 2020-08-21
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 ribonucleotide reductase transcriptional inhibitor mutant gene wxya ( wxya ) for vitamin B 2 synthetic report

Method used

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  • Ribonucleotide reductase transcription inhibitor mutant, mutant gene and its application in the preparation of vitamin b2
  • Ribonucleotide reductase transcription inhibitor mutant, mutant gene and its application in the preparation of vitamin b2
  • Ribonucleotide reductase transcription inhibitor mutant, mutant gene and its application in the preparation of vitamin b2

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1: bacterial strain CGMCC NO.16132 and BS168 ribC m Perform comparative genomic analysis

[0030] strain BS168 ribC m (For the specific construction process, please refer to Chinese patent application 201910604170.3), the genome was used as the reference genome, and the strain CGMCC NO. 16132 was sent to Jinweizhi Biotechnology Co., Ltd. for whole-genome resequencing and differential analysis to find out the effect on vitamin B production. 2 Ability-affected genes. Through sequence comparison analysis, it was found that the strain CGMCC NO. 16132 had a large number of mutations, including a total of 338 mutation sites: after mutation 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 involv...

Embodiment 2

[0032] Embodiment 2: Construct containing wxya mutant strain

[0033] by Bacillus subtilis Chromosome 168 was used as a template, primers UPnrdR-F and UPnrdR-R (including DR) were used to amplify the UPnrdR (including DR) fragment with adapter, and primers araR-F and araR-R (including DR) were used to amplify the araR with adapter ( DR) fragment; use the pC194 plasmid as a template, use primers cat-F, cat-R to amplify the cat fragment with linker; use the chromosome of strain CGMCC NO. 16132 as a template, use primer nrdR m -F, nrdR m -R amplifies nrdR with a point mutation m Fragment, wherein the mutated wxya The nucleotide sequence is shown in SEQ ID No.2, and the encoded amino acid sequence is shown in SEQ ID No.4.

[0034] Take UPnrdR fragment, cat fragment, araR fragment and nrdR m Fragment as template, use primers UPnrdR-F, nrdR m -R for fusion PCR to obtain the assembly fragment UCR-nrdR m , was detected correctly by nucleic acid electrophoresis, and purified...

Embodiment 3

[0043] Example 3: Evaluation of B vitamins in different strains 2 production capacity

[0044] 1. Strain culture conditions:

[0045] Will start strain BS168 ribC m , and engineering strain BS168 nrdR 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 OD 600 and vitamin B 2 Yield.

[0046] , OD of different strains 600 and vitamin B 2 Yield Comparison

[0047] with th...

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Abstract

The invention discloses a ribonucleotide reductase transcription inhibitor mutant, a mutant gene and its use in the preparation of vitamin B 2 in the application. Wherein the amino acid sequence of the mutant has the following mutation relative to the sequence shown in SEQ ID No.3: the 26th amino acid is replaced by H. The genetically engineered bacteria obtained by replacing the 26th amino acid encoded by the ribonucleotide reductase transcription inhibitor gene on the chromosome of Bacillus subtilis with H by site-directed mutation, which can produce vitamin B 2 The ability has been greatly improved, and it has great application and promotion value.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a mutant of ribonucleotide reductase transcription inhibitor, its genetically engineered bacteria and vitamin B 2 in the application. Background technique [0002] riboflavin aka vitamin B 2 , is a water-soluble B vitamin that most microorganisms and plants can synthesize independently, while humans and animals can only ingest it from food. Riboflavin mainly exists in the form of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) in the body, and it is used as a coenzyme or prosthetic group of flavoprotein to participate in the electron transfer and oxidation of the respiratory chain of the body tissue Reduction reaction is an essential nutrient for maintaining normal metabolism and physiological functions of the body. Riboflavin is widely used in the fields of medicine, food and feed. Due to the wide use of riboflavin, the demand for riboflavin at home and abroad i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/32C12N15/31C12N15/75C12N1/21C12P25/00C12R1/125
CPCC07K14/32C12N15/75C12P25/00
Inventor 张大伟夏苗苗刘川孙宜文
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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