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Carbon catabolism regulatory protein CcpA mutant K31A

A technology for catabolism and protein regulation, applied in recombinant DNA technology, introduction of foreign genetic material using vectors, biochemical equipment and methods, etc., can solve problems such as bacterial growth restriction and destruction of bacterial metabolic pathways

Active Publication Date: 2020-12-11
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Therefore, at present, it is generally believed in the field that simply knocking out the carbon catabolism regulatory protein will limit the growth of the bacteria, destroy the metabolic pathways of the bacteria, and often fail to obtain the expected results. With the advancement of protein engineering, the rationality of protein Retrofitting is becoming more and more popular

Method used

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  • Carbon catabolism regulatory protein CcpA mutant K31A
  • Carbon catabolism regulatory protein CcpA mutant K31A
  • Carbon catabolism regulatory protein CcpA mutant K31A

Examples

Experimental program
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Effect test

Embodiment 1

[0039] The selection of the key amino acid of embodiment 1 bacillus licheniformis CcpA protein

[0040]As a global regulatory factor, the CcpA gene of Bacillus licheniformis mainly binds to the nucleic acid site through the protein and then exerts its regulatory function. After the CcpA protein binds to the nucleic acid, the relative position of the substructure of the corresponding binding site will change to a certain extent. The amino acids that act as fulcrums in this transformation process may be located at both ends of the α-helix in the substructure of CcpA protein. At the same time, the binding of CcpA protein to nucleic acid requires the recognition of the protein and the cre site, and the amino acid that plays a major role in the recognition of the protein and the cre site may be located in the middle of the α helix, so when we select the amino acid site, it is mainly distributed in the CcpA protein α Both ends of the spiral and the middle.

Embodiment 2

[0041] The construction of embodiment 2 bacillus licheniformis CcpA protein expression vector

[0042] The Bacillus licheniformis CcpA protein genome ATCC 9945A was used as a template to amplify the CcpA gene, and the obtained gene was connected to the pET28a vector to construct the Bacillus licheniformis CcpA protein expression vector. Transform it into Escherichia coli BL21(DE3) to construct a recombinant strain.

Embodiment 3

[0043] Example 3 Site-directed Mutagenesis, Expression and Purification of Bacillus licheniformis CcpA Protein

[0044] The Bacillus licheniformis CcpA protein expression vector constructed in Example 2 was used as a template to perform site-directed mutation on the Bacillus licheniformis CcpA protein.

[0045] The primer sequences for site-directed mutagenesis are as follows:

[0046] K31A-F (SEQ ID NO.5): 5'gaaatccgaacgtcgcgccgacgacgagaaagaaggtgcttgaagccatc3';

[0047] K31A-R (SEQ ID NO. 6): 5'ttctcgtcgtcggcgcgacgttcggatttccgttcacaaccctggaaacg3'.

[0048] The CcpA protein after site-directed mutation (the amino acid sequence is shown in SEQ ID NO.2, and the nucleotide sequence of the gene encoding it is shown in SEQ ID NO.1) was heterologously expressed and purified in Escherichia coli, and the colonies were amplified by PCR. The verification diagram is as follows figure 1 As shown, the SDS-PAGE gel verification picture is as follows figure 2 As shown, it was shown that...

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Abstract

The invention discloses a carbon catabolism regulatory protein CcpA mutant K31A, and belongs to the technical field of protein engineering. A CcpA gene cloned from a bacillus licheniformis genome is mutated to obtain the carbon catabolism regulatory protein CcpA mutant K31A, the amino acid sequence of the carbon catabolism regulatory protein CcpA mutant K31A is shown as SEQ ID NO.2, and the nucleotide sequence of the gene encoding the carbon catabolism regulatory protein CcpA mutant K31A is shown as SEQ ID NO. 1. A recombinant expression vector containing the gene encoding the CcpA mutant K31Ais constructed, and the constructed expression vector is transformed into a bacillus licheniformis CcpA gene defect strain to obtain recombinant bacillus licheniformis. The obtained recombinant bacillus licheniformis can obviously change the carbon catabolism repression phenomenon caused by the existence of glucose, and the preference of the bacillus licheniformis to xylose can be reduced.

Description

technical field [0001] The invention belongs to the field of protein engineering, relates to a carbon catabolism regulating protein CcpA mutant K31A, also relates to a recombinant expression vector and a recombinant microbial cell expressing the mutant, and further relates to the effect of the mutant on Dangwood in the fermentation process of Bacillus licheniformis Effects of sugar on xylose utilization when co-existing with glucose. Background technique [0002] Bacillus licheniformis (Bacillus licheniformis) is a Gram-positive bacterium with the advantages of heat resistance, rich enzyme system, high enzyme production, moderate growth rate, protein folding, and genome-wide information disclosure. Compared with Escherichia coli, Bacillus licheniformis has the advantages of high heat resistance, low pH tolerance, and high biomass. Therefore, it is not only widely used as a host for exogenous gene expression, but also has great potential in the fermentation industry. potenti...

Claims

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Application Information

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IPC IPC(8): C07K14/32C12N15/31C12N15/75C12N1/21C12R1/10
CPCC07K14/32C12N15/75
Inventor 石贵阳李由然张玉鹏肖丰旭王瀚容张梁丁重阳徐沙顾正华
Owner JIANGNAN UNIV
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