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Method for producing riboflavin

A riboflavin and construction method technology, applied in the field of riboflavin production, can solve the problems of harmful mutation, by-produced proline or threonine in production strains, complex genetic background of production strains, etc. added effect

Active Publication Date: 2021-03-30
TONGLIAO MEIHUA BIOLOGICAL SCI TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method is random mutagenesis, and the mutation points introduced are uncertain, and often introduce more invalid mutations or even harmful mutations, making the production strains have a complex genetic background; and the newly obtained production strains produce proline or There are more threonine, etc., which is unfavorable for industrial production

Method used

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  • Method for producing riboflavin
  • Method for producing riboflavin
  • Method for producing riboflavin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1. Obtain riboflavin high-yielding bacterial strain by mutagenesis screening

[0047] With Bacillus subtilis 168 (Bacillus subtilis 168) as the original strain, the B. subtilis 168 strain was subjected to conventional mutagenesis treatment with ultraviolet 15W, 30cm, and 20min, and then mutagenized with nitrosoguanidine under the conditions of 0.4mg / mL, 36°C, 20min. Then, spread on the minimal medium containing 0.2g / L 8-azaguanine (g / L: glucose 20, ammonium sulfate 2, magnesium sulfate 0.4, calcium chloride 0.02, ferrous sulfate 0.02, disodium hydrogen phosphate 1.5, zinc sulfate 0.01, manganese sulfate 0.01, potassium dihydrogen phosphate 1.5, agar 18, pH7.0-7.2), culture at 36°C for 24 hours. Afterwards, the strain with the best growth was selected for the next round of mutagenesis, and the concentration of 8-azaguanine in the basic medium was increased. After multiple rounds of mutagenesis screening, the B. subtilis MHZ-1908-1 strain was obtained, which ...

Embodiment 2

[0049] Example 2: Construction of B. subtilis168, Δupp strain by gene seamless editing method

[0050]Using Bacillus subtilis B. subtilis168 as the starting strain, the gene scarless editing method used in the present invention is based on the two-step integration mediated by thermosensitive plasmids, through chloramphenicol positive screening and 5-fluorouracil (5-FU) reverse screening. This screening method needs to first delete the upp gene on the genome of the target bacterial strain, which encodes uracil phosphoribosyltransferase, and when upp and 5-FU exist simultaneously, it has a lethal effect on the cells. For specific principles and operation methods, please refer to the following literature: Applied Microbiology and Biotechnology, 2014, 98(21): 8963-8973. Zhang W, Gao W, Feng J, et al.

[0051] The specific construction process is as follows: using primers upp-1f / 1r, upp-2f / 2r, using the B. subtilis 168 genome as a template, using pfu DNA polymerase to amplify the u...

Embodiment 3

[0052] Example 3: Engineering strain B.subtilis 168,Δupp,ribR R97G,K110E build

[0053] Using primers ribR-1f, ribR-1r and ribR-2f, ribR-2r, and using the B. subtilis 168 genome as a template, use pfu high-fidelity DNA polymerase to amplify to obtain ribR R97G Upstream and downstream homology arms; use primers ribR-1f and ribR-2r to fuse and amplify the upstream and downstream fragments to obtain ribR R97G Fusion fragment (containing R97G mutation).

[0054] With primers ribR-1f, ribR-3r and ribR-3f, ribR-2r, with ribR R97G The fusion fragment was used as a template, and amplified using pfu high-fidelity DNA polymerase to obtain ribR R97G,K110E Upstream and downstream homology arms; use primers ribR-1f and ribR-2r to fuse and amplify the upstream and downstream fragments to obtain ribR R97G,K110E Fusion fragment (containing two point mutations R97G and K110E).

[0055] will ribR R97G,K110E The fusion fragment and the pKSU plasmid (tool vector) were subjected to SalI and ...

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Abstract

The invention belongs to the field of microbial fermentation, and discloses a method for producing riboflavin. The method for producing riboflavin comprises a construction method of a riboflavin producing strain and a method for producing riboflavin by fermenting the constructed strain. The bacillus subtilis is characterized in that at least one gene locus of purR, ribR, ribC or pyrE in a bacillussubtilis strain is mutated. The accumulation of riboflavin in the bacillus subtilis strain obtained by mutation of at least one gene locus of purR, ribR, ribC or pyrE under the fermentation conditionis remarkably increased. The bacillus subtilis is a riboflavin high-yield strain, the produced riboflavin is remarkably increased, and a foundation is laid for industrial production of the riboflavin.

Description

technical field [0001] The invention belongs to the field of microbial fermentation and discloses a method for producing riboflavin. The method for producing riboflavin in the present invention includes a method for constructing riboflavin-producing strains, and a method for producing riboflavin by fermenting the constructed strains. Background technique [0002] Riboflavin (Riboflavin), also known as vitamin B 2 , the molecular formula is C 17 h 20 o 6 N 4 , Molecular weight: 376.36, its chemical structure is as follows figure 1 shown. Riboflavin is one of the 13 essential vitamins and a member of the B vitamins. It is slightly soluble in water, soluble in sodium chloride solution, easily soluble in dilute sodium hydroxide solution, slightly soluble in ethanol, Cyclohexanol, ethyl acetate, benzyl alcohol and phenol, insoluble in ether, chloroform, acetone and benzene. Stable in neutral or acidic solution, resistant to heat and oxidation, but unstable in alkaline sol...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/75C12N1/21C12N15/54C12P25/00A23K10/18A23L29/00A61K35/742C12R1/125
CPCC12N15/75C12N15/52C12N9/1205C12P25/00C12Y207/01026A23K10/18A23L29/065A61K35/742
Inventor 吴涛胡丹常利斌龚华李岩赵津津
Owner TONGLIAO MEIHUA BIOLOGICAL SCI TECH CO LTD
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