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Bacillus subtilis adenylosuccinate synthetase mutant gene purA and applications thereof

A technology of Bacillus subtilis and adenosuccinic acid, which is applied in the field of biotechnology and molecular biology, can solve the problems such as breeding of high-yielding strains of Bacillus subtilis adenosuccinate synthase mutant gene purA riboflavin, etc. Achieve the effect of improving ability and clearing genetic background

Active Publication Date: 2014-07-30
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, for the construction of riboflavin-synthesizing engineering bacteria using Bacillus subtilis as a host, there is no report on the use of the Bacillus subtilis adenosylsuccinate synthase mutant gene purA (C725T) for the breeding of riboflavin high-yielding strains

Method used

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  • Bacillus subtilis adenylosuccinate synthetase mutant gene purA and applications thereof
  • Bacillus subtilis adenylosuccinate synthetase mutant gene purA and applications thereof
  • Bacillus subtilis adenylosuccinate synthetase mutant gene purA and applications thereof

Examples

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Embodiment 1

[0019] Example 1: Construction of the starting strain B.subtilis BUK and the basic operation plasmid pSS for screening the Bacillus subtilis system without antibiotic markers

[0020] The starting strain B. subtilis BUK 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, please refer to published document 1.

[0021] The present invention does not limit the construction method of the basic operation plasmid pSS and the selection of the resistance gene.

Embodiment 2

[0022] Embodiment 2: Construction process of engineering strain B.subtilis RC1

[0023] (1) The operation process of introducing the gene mutation ribC* (G596A) into the genome of the strain B. subtilis BUK without trace is as follows:

[0024] 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 Thermo Fast digest AatII 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 double digested with Thermo Fast digest SalI and ScaI, ligated and transformed into plasmid pSS-ribC*-F to obtain the plasmid pSS-ribC*-FB. The plasmid with the correct sequencing result was intro...

Embodiment 3

[0025] Example 3: Construction of engineering strain B. subtilis RC4 (introduction of adenosuccinate synthase mutant gene purA (C725T))

[0026] Using two pairs of primers, purA*-F-U, purA*-F-L and purA*-B-U, purA*-B-L, using the B.subtilis168 genome as a template, using KOD-plus high-fidelity DNA polymerase to amplify the size of 933bp and 922bp respectively The upstream and downstream homology arms of purA*-F and purA*-B. After the PCR fragment of purA*-F was recovered by gel cutting, it was double-digested with Thermo Fast digest NdeI and NcoI, connected and transformed into plasmid pSS to obtain plasmid pSS-purA*-F. The PCR fragment of purA*-B was recovered by cutting the gel, and then digested with Thermo Fast digest SalI and KpnI. After ligation and transformation of plasmid pSS-purA*-F, the plasmid pSS-purA*-FB was obtained. The plasmid with the correct sequencing result was introduced into B. subtilis RC1 by Spizizen transformation, and the positive clones with succes...

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Abstract

The invention discloses a Bacillus subtilis adenylosuccinate synthetase mutant gene purA and applications thereof. The sequence of the Bacillus subtilis adenylosuccinate synthetase mutant gene purA is shown in SEQ ID No.1. Engineering bacteria containing the Bacillus subtilis adenylosuccinate synthetase mutant gene purA (C725T) and built according to the invention are good in bio-safety and clear in genetic background, and the contained mutant gene purA (C725T) can significantly improve the riboflavin synthesis capacity of Bacillus subtilis, and under the condition of fermentation in a shaking flask, the level of riboflavin accumulation is increased by over 40%.

Description

technical field [0001] The invention belongs to the field of biotechnology and molecular biology, and in particular relates to the mutant gene purA (C725T) of Bacillus subtilis adenosine succinate synthetase, 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. Among the Bacillus genus, many strains including Bacillus subtilis have a reliable 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 purine metabolites. As a model strain, we have a ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/52C12N9/00C12N1/21C12P25/00C12R1/125
Inventor 王智文王光路石婷陈涛赵学明
Owner TIANJIN UNIV
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