Methionine synthetase mutant, mutant gene and application of methionine synthetase mutant and mutant gene in preparation of vitamin B12

A methionine synthetase technology, applied in the biological field, can solve problems such as metH, which does not yet have a gene encoding methionine synthase

Active Publication Date: 2020-02-21
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 methionine synthase encoding gene metH for vitamin B 12 synthetic report

Method used

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  • Methionine synthetase mutant, mutant gene and application of methionine synthetase mutant and mutant gene in preparation of vitamin B12
  • Methionine synthetase mutant, mutant gene and application of methionine synthetase mutant and mutant gene in preparation of vitamin B12
  • Methionine synthetase mutant, mutant gene and application of methionine synthetase mutant and mutant gene in preparation of vitamin B12

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0028] (2) Preparation of standard products

[0029] Configuration gradient vitamin B 12 Standards (20 mg / L, 50 mg / L, 100 mg / L, 150 mg / L).

[0030] (3) HPLC detection conditions

[0031] C18-250A column (Agilent, 4.6 mmid 9 × 250 mm, 5 µm). The mobile phase was 70% organic phase (acetonitrile) and 30% inorganic phase (sodium acetate aqueous solution), the absorption wavelength was 361 nm, the column temperature was 35 °C, the flow rate was 0.8 mL / min, and the injection volume was 20 µL.

[0032] (4) Vitamin B 12 Drawing of standard curve

[0033]Standards with different concentrations were detected by HPLC according to the above conditions, and the peak areas A-VB were drawn 12 Concentration standard curve. Taking the measured peak area A as the ordinate, vitamin B 12 The mass concentration C (mg / L) is recorded as the abscissa, and vitamin B is drawn 12 standard curve line. See Figure 4 , get the regression equation y=19.846x-80.857, R 2 =0.999, the absorbance has ...

Embodiment 1

[0034] Example 1: Determination of mutagenesis time by atmospheric room temperature plasma (ARTP), construction of mutant library and acquisition of high-yielding strains

[0035] (1) Determination of lethality

[0036] In order to obtain a relatively broad mutant library, we carried out atmospheric room temperature plasma (ARTP) mutagenesis on the chassis cell Sinorhizobium meliloti CGMCC NO.9638. First, the lethality of CGMCCNO.9638 was determined under plasma mutagenesis conditions. CGMCC NO.9638 cells (OD 600 =1) Rinse twice with 0.85% NaCl solution, then dilute to 10 with 0.85% NaCl solution 8 cells / mL suspension. Take 10uL of the suspension and spread it evenly on the iron sheet, and carry out ARTP mutagenesis. Three replicates were plated for each time point. After mutagenesis, place the iron sheet with cells in 1 mL of sterile water and vortex to wash the cells, and dilute the cell suspension 10 times to 10 -3 , Take 100 μL of the diluted cell suspension and spre...

Embodiment 2

[0046] Example 2: Comparative genome analysis of mutant strains and starting strains

[0047] The strain SM* and the starting strain CGMCC NO.9638 were sent to Jinweizhi Biotechnology Co., Ltd. for whole genome sequencing. By comparing the whole genome sequences of the two strains, it was found that a point mutation occurred in the metH gene encoding methionine synthase, and its 406th nucleotide contained a mutation. Nucleotides also contain mutations in which an A is substituted for a G. In order to verify the effect of the mutation site on vitamin B 12 Yield impact, the metH gene before and after mutation was overexpressed in the starting strain CGMCC NO.9638.

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Abstract

The invention discloses a methionine synthetase mutant, a mutant gene and an application of the methionine synthetase mutant and the mutant gene in preparation of vitamin B12. Overexpressed genetic engineering strains of a methionine synthetase gene and a mutant gene in Sinorhizobium meliloti have greatly-improved vitamin B12 producing capability, scarcely have influence on biomass during fermentation culture and have relatively high application and popularization values.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to methionine synthetase mutants, mutant genes and their use in the preparation of vitamin B 12 in the application. Background technique [0002] Vitamin B 12 (VB 12 ) is widely used in the pharmaceutical and food industries. It is also called cobalamin, which belongs to corrin compounds. It is the only vitamin compound containing metal elements and is the latest macromolecular organic compound discovered by B vitamins. Depending on the type of ligand (R group) above the corrin ring, vitamin B 12 Can be divided into: hydroxycobalamin, deoxyadenosylcobalamin and methylcobalamin. Vitamin B 12 Participated in a large number of biochemical reactions including DNA synthesis and regulation, fatty acid synthesis, amino acid metabolism and capacity generation. [0003] due to vitamin B 12 The molecular structure is complex, and chemical synthesis requires a lot of manpower a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/74C12P19/42C12N13/00C12N1/20C12R1/41
CPCC12N9/1085C12N13/00C12N15/74C12P19/42C12Y205/01006C12N1/205C12R2001/41
Inventor 张大伟董会娜
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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