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A carbon catabolism regulatory protein CCPA mutant K31A

A technology for catabolism and regulation of proteins, which is applied in the field of carbon catabolism regulation protein CcpA mutant K31A, recombinant expression vectors and recombinant microbial cells, which can solve the problems of destroying bacterial metabolic pathways and restricted bacterial growth, so as to reduce preference sex, altering the effect of carbon catabolism repression

Active Publication Date: 2022-02-01
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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  • A carbon catabolism regulatory protein CCPA mutant K31A
  • A carbon catabolism regulatory protein CCPA mutant K31A
  • A carbon catabolism regulatory protein CCPA mutant K31A

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Choice of key amino acids of CCPA protein in Bacillus bacillus

[0040]The CCPA gene of the Jacket Bacillus is mainly combined by a global regulatory factor mainly by protein and a nucleic acid site, and then exerts its regulation function, the relative position of the sub-structure of the CCPA protein and the sub-structure of its corresponding binding site after the nucleic acid is changed. Amino acid acting in this transform process may be located at both ends of the α spiral in the CCPA protein sub-structure. At the same time, the CCPA protein and the nucleic acid are identified by the protein and the CRE site, and the amino acid in which the primary action thereof in the protein and the CRE site may be located in the middle of the α spiral, so we are mainly distributed at CCPA protein when selecting the amino acid sites. Α The two ends of the spiral and the middle.

Embodiment 2

[0041] Example 2 Construction of CCPA protein expression vector

[0042] The CCPA gene was amplified by the CCPA protein genome of the Jacket Bacillus, and the CCPA gene was amplified, and the resulting gene was attached to the PET28a vector, and the Bacillus CCPA protein expression vector was constructed. It was converted to E. coli BL21 (DE3) to construct recombinant strains.

Embodiment 3

[0043] Example 3 CCPA protein at CCPA protein and expression and purification

[0044] The CCPA protein expression vector constructed in Example 2 was a template to a fixed point mutation of the CCPA protein of the Jacket Bacillus underclothes.

[0045] The order of the fixed-point mutant primer is as follows:

[0046] K31A-F (SEQ ID No.5): 5'GaAatcggaacgtcgccccgcgcgagaagaagaaggtgcttgaagccc3 ';

[0047] K31A-R (SEQ ID No.6): 5'tctcgtcgggcgcgcgttcgggttccgttcacaaccctggAAACG3 '.

[0048] The CCPA protein after the fixed point mutation (amino acid sequence, such as SEQ ID NO.2, encoding the gene nucleotide sequence, such as SEQ ID NO.1), is subjected to heterologous expression and purification, colony PCR expansion Introduction figure 1 As shown, SDS-PAGE gel verification figure 2 As shown, it indicates that the CCPA protein prototype of the Jacket Bacillus CCPA protein is successfully introduced into E. coli and successfully expressed in E. coli.

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Abstract

The invention discloses a carbon catabolism regulating protein CcpA mutant K31A, which belongs to the technical field of protein engineering. The present invention mutates the CcpA gene cloned from the Bacillus licheniformis genome to obtain a carbon catabolism regulatory protein CcpA mutant K31A, whose amino acid sequence is shown in SEQ ID NO.2, and the gene nucleotide sequence encoding it is shown in Shown in SEQ ID NO.1. The present invention constructs a recombinant expression vector containing the above-mentioned CcpA mutant K31A gene, and transforms the constructed expression vector into a CcpA gene-deficient strain of Bacillus licheniformis to obtain the recombinant Bacillus licheniformis. The obtained recombinant bacillus licheniformis can obviously change the phenomenon of carbon catabolism repression caused by the presence of glucose, and can reduce the preference of the bacillus licheniformis to xylose.

Description

Technical field [0001] The present invention belongs to the field of protein engineering, and involves a carbon decomposition metabolic regulatory protein CCPA mutant K31A, and further relates to recombinant expression vectors expressing the mutant and recombinant microbial cells, and further relates to the mutant. Effects of Sugar and Glucose Co - co - co - co - co - co - co - co - co - co - coordination. Background technique [0002] Bacillus licheniformis is a Gram-positive bacteria, which has the advantages of heat resistance, rich enzyme, high enzyme, moderate growth rate, moderate protein fold, and all genome information disclosure. Compared with E. coli, the bacillus undergarment has high heat resistance, low pH tolerance, high biomass, not only used for expression hosts of exogenous genes, but also has huge in the fermentation industry. potential. [0003] As a high-quality carbon source, xylose can be obtained by hydrolysis of lignocellulose, with the current environment...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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