Bacillus subtilis-coded PRPP (Phosphoribosyl Pyrophosphate) transamidase mutant gene pruF and application thereof

A technology of Bacillus subtilis and mutant gene, applied in the fields of biotechnology and molecular biology, can solve the problem that there is no Bacillus subtilis riboflavin high-yielding strain and the like, and achieve the improvement of riboflavin accumulation level, ability and genetic background. clear effect

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

[0005] At present, there is no report on the breeding of riboflavin high-yielding strains with the mutant gene purF encoding PRPP transamidase in Bacillus subtilis

Method used

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  • Bacillus subtilis-coded PRPP (Phosphoribosyl Pyrophosphate) transamidase mutant gene pruF and application thereof
  • Bacillus subtilis-coded PRPP (Phosphoribosyl Pyrophosphate) transamidase mutant gene pruF and application thereof
  • Bacillus subtilis-coded PRPP (Phosphoribosyl Pyrophosphate) transamidase mutant gene pruF and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0028] Embodiment 1: Construction of basic operation plasmid pSS

[0029] PCR reaction was used to obtain the cat gene using the pC194 (source: Bacillus Genetic Stock Center, BGSC, http: / / www.bgsc.org / ) plasmid as a template, using the upstream and downstream primers pSS-P1 and pSS-P2, and the genome of B.subtilis168 as The template uses the upstream and downstream primers pSS-P3 and pSS-P4 to obtain the upp gene, and then uses the above two fragments as a template to obtain the recombinant fragment Cat-Upp using the upstream and downstream primers pSS-P1 and pSS-P4 by fusion PCR reaction. The recombinant fragment and the pUC18 (universal vector) plasmid are subjected to enzyme digestion, enzyme connection, transformation, verification, etc., to obtain the basic operation plasmid pSS, see figure 1 .

[0030] The present invention does not limit the construction method of the basic operation plasmid pSS and the selection of the resistance gene.

Embodiment 2

[0031] Example 2: construction of purF point mutation introduced into plasmid pSS-purF*-FB

[0032] Using a pair of primers purF-F-U and purF-F-L, using the B.subtilis168 genome as a template, using KOD-plus high-fidelity DNA polymerase to amplify the upstream homology arm purF*-F with a size of 836bp, the lower primer purF- The mutation point D293V was introduced in F-L. The PCR fragment of purF*-F was recovered by gel cutting, and then digested with Thermo Fast digest BglII and XhoI. After ligation and transformation of plasmid pSS, plasmid pSS-purF*-F was obtained.

[0033]Using a pair of primers purF-B-Fsn-1 and purF-B-Fsn-2, using the B. subtilis168 genome as a template, using KOD-plus high-fidelity DNA polymerase to amplify fragment A with a size of 345bp, in which the upper primer The mutation point K316Q was introduced into purF-B-Fsn-1, and the mutation point S400W was introduced into the lower primer purF-B-Fsn-2. Using a pair of primers purF-B-Fsn-3 and purF-B-Fsn...

Embodiment 3

[0034] Example 3: Construction of Bacillus subtilis strain B.subtilis BUK

[0035] The starting strain B. subtilis BUK used in the present invention is derived from B. subtilis 168, and the detailed construction process can refer to published document 1.

[0036] The bacterial strain has the ability to quickly prepare competent cells under induction conditions, and simultaneously has a high absorption capacity of exogenous DNA.

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Abstract

The invention discloses a bacillus subtilis-coded PRPP (Phosphoribosyl Pyrophosphate) transamidase mutant gene pruF and an application thereof. The bacillus subtilis-coded PRPP transamidase mutant gene pruF sequence is shown as SEQ ID No.1. An engineering bacterium which is established and contains the bacillus subtilis-coded PRPP transamidase mutant gene pruF is biologically safe and clear in genetic background, so that the capacity of the bacillus subtilis which synthesizes riboflavin can be greatly improved, and the accumulation level of riboflavin can be improved by over 20%.

Description

technical field [0001] The invention belongs to the field of biotechnology and molecular biology, and in particular relates to a Bacillus subtilis-encoded PRPP transamidase mutant gene purF and an amino acid sequence encoded by the mutant gene, including an engineering bacterium comprising a Bacillus subtilis-encoded PRPP transamidase mutant gene purF And the application of the bacteria in producing riboflavin. 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 found that the mutant strains of Bacillus subtilis can over-synthesize a series of purine pathway metabolic intermediates such as folic acid, adenosine, inosine, guanosine, riboflavin or derivative metabolites of this pathway, ...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N9/10C12N1/21C12P25/00C12R1/125
Inventor 王智文石婷王永成陈涛赵学明
Owner TIANJIN UNIV
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