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Two-component regulatory system mutant gene yvrh of Bacillus subtilis and its application

A Bacillus subtilis and mutant gene technology, which is applied in the fields of biotechnology and molecular biology, can solve the problems of high-yielding strains of mutant gene riboflavin that do not yet have a two-component regulation system of Bacillus subtilis, and achieves improved capacity and increased accumulation. Level, clear effect of genetic background

Active Publication Date: 2016-01-20
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is no report on the use of the two-component regulatory system mutant gene yvrH (G665A) in Bacillus subtilis for breeding strains with high riboflavin production

Method used

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  • Two-component regulatory system mutant gene yvrh of Bacillus subtilis and its application
  • Two-component regulatory system mutant gene yvrh of Bacillus subtilis and its application
  • Two-component regulatory system mutant gene yvrh of Bacillus subtilis and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0020] Example 1: Construction of the starting strain B.subtilisBUK and the basic operation plasmid pSS for screening the Bacillus subtilis system without antibiotic markers

[0021] The starting strain B.subtilisBUK used in the present invention is derived from B.subtilis168, and the basic operation plasmid pSS is derived from pUC18. For the detailed construction process of the two, refer to published document 1.

[0022] The present invention does not limit the construction method of the basic operation plasmid pSS and the selection of the resistance gene.

Embodiment 2

[0023] Embodiment 2: Construction process of engineering strains B.subtilisRC1 and B.subtilisRC2

[0024] (1) The procedure for introducing the gene mutation ribC* (G596A) into the genome of strain B.subtilisBUK without trace is as follows:

[0025] Using ribC*-F-U, ribC*-F-L and ribC*-B-U, ribC*-B-L two pairs of primers, using the B. subtilis168 genome as a template, using KOD-plus high-fidelity DNA polymerase to amplify the size of 894bp and 928bp respectively The upstream and downstream homology arms of ribC*-F and ribC*-B. After the PCR fragment of ribC*-F was recovered by gel cutting, it was digested with ThermoFastdigestAatII and XhoI, connected and transformed into plasmid pSS to obtain plasmid pSS-ribC*-F. After the PCR fragment of ribC*-B was recovered by gel cutting, it was digested with ThermoFastdigestSalI and ScaI, connected and transformed into plasmid pSS-ribC*-F to obtain the plasmid pSS-ribC*-FB. The plasmid with the correct sequencing result was introduced ...

Embodiment 3

[0028] Example 3: Construction of engineering strain B.subtilisRC3 (introduction of Bacillus subtilis two-component regulatory system mutant gene yvrH (G665A))

[0029]Using two pairs of primers yvrH*-F-U, yvrH*-F-L and yvrH*-B-U, yvrH*-B-L, using the B.subtilis168 genome as a template, using KOD-plus high-fidelity DNA polymerase to amplify the size of 1067bp and 849bp respectively The upstream and downstream homology arms of yvrH*-F and yvrH*-B. After the PCR fragment of yvrH*-F was recovered by cutting the gel, it was double digested with ThermoFastdigestAatII and XhoI, connected and transformed into plasmid pSS to obtain plasmid pSS-yvrH*-F. After the PCR fragment of yvrH*-B was recovered by cutting the gel, it was digested with ThermoFastdigestSalI and KpnI, connected and transformed into plasmid pSS-yvrH*-F to obtain plasmid pSS-yvrH*-FB. The plasmid with the correct sequencing result was transformed into Bacillus subtilis B.subtilisRC2 by Spizizen, and the positive clon...

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Abstract

The invention discloses a two-component regulatory system mutant gene yvrH (G665A) of bacillus subtilis and application thereof. The two-component regulatory system mutant gene yvrH (G665A) of the bacillus subtilis is shown in SEQ ID No.1. The engineering bacteria containing the two-component regulatory system mutant gene yvrH (G665A) of bacillus subtilis built by the invention have biosafety and clear genetic backgrounds, the ability of the bacillus subtilis to synthesize riboflavin can be greatly improved by the contained mutant gene yvrH (G665A), and the riboflavin accumulation level can be improved by over 15% under a shake flask fermentation condition.

Description

technical field [0001] The invention belongs to the field of biotechnology and molecular biology, and specifically relates to a mutant gene yvrH (G665A) of a two-component regulatory system of Bacillus subtilis, the amino acid sequence encoded by the gene and its application. Background technique [0002] Genetically engineered bacteria with bacteria as host bacteria have the advantages of short fermentation cycle, simple raw material requirements, and mature genetic engineering technology. Within the Bacillus genus, many strains, including Bacillus subtilis, have a proven safety profile. Traditional strain breeding has found that the mutant strains of Bacillus subtilis can oversynthesize a series of purine pathway metabolic intermediates such as folic acid, adenosine, inosine, guanosine, riboflavin or derivative metabolites of this pathway, and are currently becoming a high-yield breeding method. Important starting strains for nucleoside metabolites. As a model strain, we...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/31C07K14/32C12N1/21C12P25/00C12R1/125
Inventor 王智文王光路石婷陈涛赵学明
Owner TIANJIN UNIV
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