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Bacillus subtilis capable of stably expressing chitobiose deacetylase as well as construction method and application of bacillus subtilis

A technology of Bacillus subtilis and deacetylase, which is applied in the field of genetic engineering, can solve the problems of long fermentation period of Bacillus subtilis, unstable cell production performance, and high cost investment, and achieve the simple and easy construction method, shorten the fermentation period, and protect the environment The effect of little pollution

Pending Publication Date: 2021-10-22
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In order to improve the current situation that heterologous proteins are easily lost in the subculture process when they are freely expressed, and the production performance of cells is unstable, the present invention provides a recombinant engineering strain that stably and efficiently expresses chitobiose deacetylase Dac continuously and efficiently through genome integration and Its construction method; in order to solve the problem of long fermentation period of Bacillus subtilis and high investment in industrial production costs, the present invention shortens the fermentation period by means of CRISPER / Cpf1 gene editing, and provides a stable expression of chitobiose deacetylase Dac in advance. Recombinant engineering strain and construction method thereof

Method used

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  • Bacillus subtilis capable of stably expressing chitobiose deacetylase as well as construction method and application of bacillus subtilis
  • Bacillus subtilis capable of stably expressing chitobiose deacetylase as well as construction method and application of bacillus subtilis
  • Bacillus subtilis capable of stably expressing chitobiose deacetylase as well as construction method and application of bacillus subtilis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Example 1: Bacillus subtilis chassis cell reconstitution

[0086] Specific steps are as follows:

[0087] CRISPR editing is realized through the DNA endonuclease activity of the Cas protein Cpf1. Under the guidance of the guide RNA (crRNA), the Cpf1 protein will cut at a specific site in the genome, resulting in a double-strand break (the application schematic diagram of the CRISPER / Cpf1 system is shown in figure 1 shown).

[0088] 1. Construction of crRNA vector

[0089] Use the crRNA online design website CRISPR-DT to design crRNA with high specificity to reduce the occurrence of off-target phenomena. The primers listed in Table 1 are used to amplify the crRNA fragments of genes comA, sigD, and lytC to be knocked out (the accession numbers of the lytC, sigD, and comA genes in NCBI are respectively: 936777, 938482, and 937179), and the sequence is as SEQ ID NO. 2 to 4, and then carried out golden gate assembly with the vector pcrF17NM, and transformed into Escherich...

Embodiment 2

[0106] Example 2: Preparation of chitobiose deacetylase Dac by free expression of heterologous protein

[0107] 1. Preparation of Competent Host Cells

[0108] The Bacillus subtilis host cells Bacillus subtilis C6C, Bacillus subtilis C6D, and Bacillus subtilis C6A obtained in Example 1 were prepared as competent cells by referring to the method for preparing Cas protein Cpf1 competent cells in Step 3 of Example 1.

[0109] 2. Construction of recombinant vector

[0110] (1) In pP43NMKmut-C 4 On the basis of -yncM-Dac, the amino acid sequence is NprB as shown in SEQ ID NO.5 5 The signal peptide replaced the yncM signal peptide, and the RBS sequence GTAAGAGAGGAATGTACAC on the pP43NMK vector was mutated to R1~R9, wherein the sequences of R1~R9 are shown in Table 3, and the sequences of the primers involved are shown in Table 4:

[0111] Table 3: RBS sequence after mutation

[0112]

[0113] Table 4: Primer sequences

[0114]

[0115] Perform PCR amplification to obtain ...

Embodiment 3

[0132] Example 3: Construction of Chitobiose Deacetylase Dac Expression Cassette

[0133] Specific steps are as follows:

[0134] Plasmid pP43NMKmut-C 4 -NprB 5 -R 1 -Dac is used as a template, and Dac-F and Dac-R are used for PCR amplification.

[0135] Chitobiose deacetylase Dac gene upstream primer Dac-F:

[0136] 5'-TGATAGGTGGTATGTTTTCGCTTGAAC-3';

[0137] Chitobiose deacetylase Dac gene downstream primer Dac-R:

[0138] 5'-TCAGTGATGGTGATGGTGATGGTG-3'.

[0139] A chitobiose deacetylase Dac integrated expression cassette with a nucleotide sequence as shown in SEQ ID NO.1 was prepared.

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Abstract

The invention discloses bacillus subtilis capable of stably expressing chitobiose deacetylase as well as a construction method and an application of the bacillus subtilis, and belongs to the technical field of genetic engineering. According to the invention, integration sites are screened, the integration copy number is increased, and a food safety level engineering strain capable of stably and efficiently expressing the chitobiose deacetylase Dac is constructed. Compared with the prior art, the extracellular enzyme activity of the chitobiose deacetylase is greatly improved, and the fermentation period is greatly shortened. The conversion method is simple and easy to implement, mild in condition, efficient, environmentally friendly and high in product yield. The method is beneficial to solving the problems of serious pollution, low yield and the like in the traditional synthesis method, and is easy to control and beneficial to popularization and application.

Description

technical field [0001] The invention relates to a bacillus subtilis stably expressing chitobiose deacetylase and its construction method and application, belonging to the technical field of genetic engineering. Background technique [0002] Chitobiose deacetylase Dac is derived from the extreme thermophilic archaea Pyrococcus horikoshii, which has high catalytic activity on acetylglucosamine monomer, compared with chemical hydrolysis and microbial catalysis to produce glucosamine GlcN, enzymatic catalysis The invention has the advantages of rich source of raw materials, low price, simple production process and the like. The establishment of an efficient and stable expression system for chitobiose deacetylase Dac can facilitate the one-step production of glucosamine GlcN. [0003] Glucosamine GlcN is a hexosamine sugar, as a functional monosaccharide involved in the construction of human tissues and cell membranes, it can be applied in many fields. In terms of immune regula...

Claims

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Application Information

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IPC IPC(8): C12N9/80C12N15/55C12N1/21C12N15/75C12P19/26C12R1/125
CPCC12N9/80C12N15/75C12P19/26
Inventor 刘龙陈坚吕雪芹堵国成李江华刘延峰毛馨竹
Owner JIANGNAN UNIV
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