Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

A method for constructing an integrated high-efficiency expression of acetaldehyde dehydrogenase Bacillus subtilis

A technology of acetaldehyde dehydrogenase and Bacillus subtilis, which is applied in the construction field of Bacillus subtilis, can solve the problems of high cost and difficulty in large-scale production, and achieve the effect of increasing yield and efficient secretion expression

Active Publication Date: 2022-03-25
NANJING AGRICULTURAL UNIVERSITY +1
View PDF16 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Most acetaldehyde dehydrogenase is extracted from animal liver, pancreas or microorganisms, the cost is high and it is difficult to achieve large-scale production

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
  • A method for constructing an integrated high-efficiency expression of acetaldehyde dehydrogenase Bacillus subtilis
  • A method for constructing an integrated high-efficiency expression of acetaldehyde dehydrogenase Bacillus subtilis
  • A method for constructing an integrated high-efficiency expression of acetaldehyde dehydrogenase Bacillus subtilis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Construction of integrated acetaldehyde dehydrogenase secretion expression vector

[0034] The integrative plasmid can be any plasmid that can replicate in Bacillus subtilis and Escherichia coli and can be integrated into the chromosome by homologous recombination. The integration site may be the position of amylase (amyE), xylanase (xylA) and any other gene not necessary for the growth of Bacillus subtilis in Bacillus subtilis. The promoter contained in the integrating plasmid can be P amyQ ,P amyE ,P amyL ,P aprE ,P xylA or P glv Any one of or other promoters, and the signal peptide can be any one of SPaprE, SPchiA, SPwapA, SPpbpA or SPyqzG or other signal peptides. In this embodiment, the integration plasmid pCBS is used, the integration site is the amyE gene, and the promoter P amyL -P amyQ -P cry3A Taking the signal peptide SPchiA as an example to describe the construction process of the integrated plasmid, the principles and processes of other ...

Embodiment 2

[0050] Example 2: Construction of Bacillus subtilis with integrated secretory expression of acetaldehyde dehydrogenase

[0051] Transformation of the recombinant plasmid: transform the recombinant plasmid pCBS / istALDH into Bacillus subtilis 168 chemically competent cells, spread the LB resistance plate (containing 5 μg / mL erythromycin), culture overnight at 37°C, and verify the transformants by PCR.

[0052] Screening of recombinant strains: pCBS plasmid is a temperature-sensitive shuttle plasmid for Escherichia coli and Bacillus subtilis, and contains a temperature-sensitive replication origin site for Gram-positive bacteria. At 37°C, autonomous replication can be carried out in the host bacteria, but when the temperature rises to 42°C, autonomous replication cannot be carried out.

[0053] The Bacillus subtilis containing the recombinant plasmid was cultured at 42°C and 180rpm for 24 hours, then spread on an LB plate, cultured at 42°C to obtain a single colony, and induced t...

Embodiment 3

[0058] Example 3: Construction of Bacillus subtilis overexpressing CsaA strain (CsaA gene GenBank: CP041757 REGION: 2078810---2079142, 333bp)

[0059] Using the genomic DNA of Bacillus subtilis 168 as a template, PCR amplification was performed with primers 9 and 10 (underlined restriction sites), and the PCR product (CsaA, 333bp) was recovered. Add A reaction to the recovered product, connect it to pMD19T vector (Takara Company) with T4 ligase, transform Escherichia coli competent cells, and obtain recombinant plasmid pMD19T / csaA (Mlu I-Sal I). Digest pMD19T / csaA (Mlu I-Sal I) and expression plasmid pBE (purchased from Takara, Cat#3380) with restriction endonucleases Mlu I and Sal I, purify the digested product, and use T4 ligase The linearized pBE vector was connected to the csaA fragment, transformed into Escherichia coli competent cells, and the sequenced correct plasmid was named pBE / csaA ( Figure 4 , Figure 6 a&c, the verified fragment is csaA, 333bp).

[0060] Tran...

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 relates to a method for constructing a Bacillus subtilis with integrated high-efficiency secretion and expression of acetaldehyde dehydrogenase, belonging to the field of biotechnology. The invention realizes the secretion and expression of the acetaldehyde dehydrogenase in the Bacillus subtilis through the integrated plasmid, and the recombinant strain has broad application prospects in the field of acetaldehyde detoxification. The invention greatly improves the yield of acetaldehyde dehydrogenase in the recombinant bacillus subtilis by co-expressing molecular chaperones PrsA and CsaA.

Description

1. Technical field [0001] The invention relates to a method for constructing a Bacillus subtilis with integrated high-efficiency secretion and expression of acetaldehyde dehydrogenase, belonging to the field of biotechnology. 2. Background technology [0002] The acetaldehyde dehydrogenase superfamily comprises a series of distinct enzymes that are widely distributed in nature, represented in all three taxonomic domains (archaebacteria, eubacteria, and eukaryotes), and have been present throughout evolutionary history to important role. Members of the aldehyde dehydrogenase family metabolize physiologically and pathophysiologically relevant aldehydes, preventing the accumulation of endogenous and / or exogenous toxic aldehydes that could adversely affect cellular homeostasis and biological function. [0003] Aldehydes are widely found in nature, and acetaldehyde is found in food, tobacco smoke and beverages. Most aldehydes are toxic, such as acetaldehyde can be mutagenic and...

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): C12N1/21C12N15/75C12N15/53C12R1/125
CPCC12N9/0008C12N15/75C12Y102/0101
Inventor 陆兆新卢静李金良吕凤霞别小妹赵海珍
Owner NANJING AGRICULTURAL UNIVERSITY
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com