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Bacillus subtilis strain capable of efficiently expressing exogenous secretory proteinase

A Bacillus subtilis and exogenous protein technology, applied in the field of genetic engineering, can solve the problems of high exocrine protease concentration and limited application, and achieve the effect of broad development prospects

Active Publication Date: 2014-10-01
NANJING BESTZYME BIO ENG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The natural strains of Bacillus subtilis have a high concentration of exocrine proteases, which greatly limits its application in fermentation engineering, especially some enzymes sensitive to proteases

Method used

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  • Bacillus subtilis strain capable of efficiently expressing exogenous secretory proteinase
  • Bacillus subtilis strain capable of efficiently expressing exogenous secretory proteinase
  • Bacillus subtilis strain capable of efficiently expressing exogenous secretory proteinase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1. Construction of pYF‐tsDE plasmid

[0040] pYF‐tsDE ( figure 1) is an Escherichia coli / Bacillus shuttle plasmid designed by the inventor. Its composition mainly includes a heat-sensitive replication origin with replication activity at 30°C and an erythromycin resistance gene. The concentration of erythromycin resistance in Escherichia coli is 300ug / ml; the concentration in Bacillus subtilis is 5ug / ml. At 37°C, the origin of replication of the plasmid cannot replicate so that the plasmid is integrated into the host genome through the designed site and screened by the erythromycin resistance gene. The construction of the pYF-tsDE plasmid is described as follows:

[0041] pUC57‐KS‐erm (plasmid constructed by Genscript) ( figure 2 , see SEQ ID NO.9 for the sequence) after digestion with BglII, the 3.8 kbp fragment was recovered and then self-ligated with T4 ligase (provided by NEB). The resulting 3.8kbp plasmid was named pYF-tsDE. This plasmid was trans...

Embodiment 2

[0042] Embodiment 2. Construction of a Bacillus subtilis strain lacking exogenous secretory protease and sporulation

[0043] The extracellular protease activity of Bacillus subtilis itself is unfavorable to the secretion of heterologous enzymes. Two extracellular proteases that have been identified: Bacillus subtilis alkaline protease aprE and Bacillus subtilis neutral metalloprotease nprE constitute 85% of the extracellular protease activity of Bacillus subtilis. In addition, spores can form dormant cells during fermentation which will result in a double reduction in production efficiency. The sigF gene encodes the sigma-F factor that controls sporulation, which plays a key role in directing the transcription of RNA polymerase and the specificity of the expression product in sporulation.

[0044] In the present invention, the deletions of the above three genes are inserted into the target gene through homologous insertion by a single cross-over Campbell-type mechanism in a...

Embodiment 3

[0097] Embodiment 3. Construction of pullulanase-producing strains

[0098] 3.1 Construction of pullulanase expression framework

[0099] A typical pullulanase expression cassette consists of the following parts:

[0100] (1) Two to three tandem natural promoters (natural promoters from B. subtilis, B. licheniformis and B. amyloliquefaciens can be selected); the sequence is SEQ ID NO.5;

[0101] (2) Synthetic ribosome binding site sequence, see SEQ ID NO.6;

[0102] (3) The expression framework inserts a strong natural signal peptide sequence upstream of the start codon of the pullulanase coding gene, see SEQ ID NO.8;

[0103] (4) The expression framework pullulanase gene is derived from B.acidopulluticus, see SEQ ID NO.1;

[0104] (5) The expression framework contains a synthetic termination sequence, see SEQ ID NO.7;

[0105] The synthesis of the above sequences was completed by Genscript Company, and the above sequences were sequentially and seamlessly concatenated to ob...

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Abstract

The invention discloses a bacillus subtilis strain capable of efficiently expressing an exogenous secretory proteinase. A bacillus subtilis host bacteria capable of expressing exogenous protein is disclosed. A gene apr of a bacillus subtilis alkaline protease, and a gene npr and a spore forming gene spoIIAC of a bacillus subtilis neutral metalloproteinase are knocked out, wherein the bacillus subtilis alkaline protease and the bacillus subtilis neutral metalloproteinase are two exocrine proteinases in the bacillus subtilis host bacteria. The obtained bacillus subtilis host bacteria can be used for high-yield exogenous proteinases by genetic engineering manners. Based on this, a genetically engineered bacterium secreting pullulanase is constructed and can produce the pullulanase with a high yield. The genetically engineered bacterium secreting has a wide development prospect in industrial production by fermentation industry research.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to a bacillus subtilis capable of highly expressing exogenous secretory protease. Background technique [0002] Bacillus subtilis is an extremely important starting strain in bioengineering and enzyme preparation industry. Today, its physical and chemical properties are known, and the genetic modification method is relatively mature; in addition, it is a non-pathogenic strain recognized by the US FDA and can be widely used in the fermentation production of enzyme preparations related to the food industry. The natural strains of Bacillus subtilis have a high concentration of exocrine proteases, which greatly limits its application in fermentation engineering, especially some enzymes sensitive to proteases. In the present invention, a strain of Bacillus subtilis deficient in exogenous secreted protease is constructed by means of genetic engineering. In addition, in order to adapt to l...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/75C12R1/125
Inventor 范岩陆亚敏杜秀贞杜华东付奇乔宾福
Owner NANJING BESTZYME BIO ENG CO LTD
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