Mutant gene purf encoding prpp transamidase in Bacillus subtilis and its application

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: 2016-06-29
TIANJIN UNIV
View PDF4 Cites 1 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Mutant gene purf encoding prpp transamidase in Bacillus subtilis and its application
  • Mutant gene purf encoding prpp transamidase in Bacillus subtilis and its application
  • Mutant gene purf encoding prpp transamidase in Bacillus subtilis and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0029] Use PCR reaction to use pC194 (source: BacillusGeneticStockCenter, BGSC, http: / / www.bgsc.org / ) plasmid as template, use upstream and downstream primers pSS-P1 and pSS-P2 to obtain cat gene, and use B.subtilis168 genome as template The downstream primers pSS-P3 and pSS-P4 were used to obtain the upp gene, and then the above two fragments were used as templates 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. After the PCR fragment of purF*-F was recovered by gel cutting, it was digested with ThermoFastdigestBglII and XhoI, connected and transformed into plasmid pSS to obtain plasmid pSS-purF*-F.

[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-4, using...

Embodiment 3

[0034] Embodiment 3: Construction of Bacillus subtilis system starting bacterial strain B.subtilisBUK

[0035] The starting strain B.subtilisBUK used in the present invention is derived from B.subtilis168, 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.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

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. Within the Bacillus genus, many strains, including Bacillus subtilis, have a proven 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Patents(China)
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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap